For antigen retrieval, pretreatment was performed by microwave he

For antigen retrieval, pretreatment was performed by microwave heating in 1 mmol/L sodium citrate puffer (30 minutes, 600 W, pH 6.0). ABT888 Incubation of each one series with anti-DAPK (mouse monoclonal antibody, dilution 1:500, BD Transduction Laboratories, Heidelberg, Germany), anti-p-p38 MAPK (rabbit monoclonal antibody, 1:100, Cell Signaling Technology, Boston, MA), and anti-M30 CytoDeath (mouse monoclonal antibody dilution 1:75, Roche Diagnostics, Basel, Switzerland) was conducted at room temperature for 12 hours and followed by PBS-washing. Positive immunohistochemical reactions were revealed using the iVIEW DAB Detection Kit (Ventana, Germany) as chromogen substrate. Specimens were counterstained with hematoxylin and mounted with DEPEX. Samples were examined by two different reviewers blinded to other data.

All aspects of this study were reviewed and approved by the Ethics Committee of the Medical Faculty of Magdeburg and all patients provided informed consent. Semiquantitative Assessment of Immunohistochemical Protein Expression and Apoptosis For DAPK and p-p38 protein expression, cytoplasmic staining intensity, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (1 = <10%; 2 = 10 to 50%; 3 = 51 to 80%; 4 = >80%) were scored semiquantitatively, resulting in an immunoreactive score (staining intensity �� positive cells) with a possible maximum of 12 points. For DAPK, an immunoreactive score of 0 to 3 points was considered as ��low expressing�� compared with samples with an immunoreactive score of 4 to 12 points, which where referred to ��high expressing.

�� Apoptosis was determined as a percentage of M30-labeled tumor cells and scored as 0 (<1%), 1 (1% to 5%), 2 (5% to 10%), 3 (10% to 15%), or 4 (>15%). Statistical Analysis Data are presented as the means of triplicates from three separate experiments �� SEM. Statistical comparisons of the amounts of cytokines and immunohistochemical protein expression between various subgroups were performed by Student��s t-test. The level of statistical significance was set at P < 0.05. Results Macrophage-Mediated Batimastat Killing of HCT116 Tumor Cells and Up-Regulation of DAPK Expression In our previous studies, DAPK protein expression in colorectal tumor cells and their tumor-associated macrophages was found to be highly correlated. Furthermore, the increase in the DAPK level was associated with higher apoptosis rate in colorectal tumor cells.19 To determine whether tumor-associated macrophages and the secretion of their cytotoxic mediators might induce apoptosis in these tumor cells, we used a cell culture assay to simulate the events that might occur in the in vivo tumor setting.

Hoffmann-La Roche, Basel,

Hoffmann-La Roche, Basel, Switzerland) according to the manufacturer’s instructions. Overnight fasting blood glucose was measured using the Ascensia Contour glucometer (Bayer HealthCare, IN). FFAs were measured in mouse serum using the Half Micro Test (Roche Diagnostics, Mannheim, Germany). Hormone measurements were also taken at 32 weeks of age. Fasting serum insulin was measured using the ultrasensitive mouse insulin ELISA kit from Mercodia (Uppsala, Sweden) according to the manufacturer’s instructions. Fasting serum leptin and adiponectin/Acrp30 were measured by ELISA using commercial assay kits (MRP300 mouse Adiponectin/Acrp30; MOB00 mouse leptin, R and D Systems, Minneapolis, MN). Fasting serum Retinol Binding Protein 4 (RBP4) was measured using the Dual mouse/rat RBP4 ELISA kit (RB0642EK; AdipoGen, Seoul, Korea).

Histology and Oil-Red-O staining of murine liver and adipose tissue Liver and visceral adipose tissues were removed and weighed. Formalin-fixed, paraffin-embedded liver and adipose tissue from eight of the 32-week-old male mice were processed, and 4-��m-thick serial sections were cut and stained with Oil-Red-O or hematoxylin and eosin (HE) for lipid analysis according to standard pathology laboratory procedures. After mounting with glycerol gelatin, images were captured using Axio Vision Rel4.5 software (Carl Zeiss). For measurement of adipocyte cell areas, images were converted to TIFF files for analysis using the Discovery SeriesTM Quantity One? 1-D analysis software (Bio-Rad Laboratories).

Hepatic TG quantitation Levels of mouse liver TG were quantified using the Triglyceride Determination Kit TRO100 with appropriate TG standards (Sigma-Aldrich, St. Louis, MO). Frozen liver samples from 32-week-old mice were first powdered under liquid nitrogen and 120 mg of the frozen liver powder was weighed into 2 ml of chloroform:methanol mix (2:1, v/v) and incubated for 2 h at room temperature with occasional shaking. Following the addition of 0.2 volumes of water, vortexing, and centrifuging at 2,500 g, the lower phase containing the lipids was collected and dried under vacuum in a rotary evaporator for 5�C6 h. The dried pellets were resuspended in the reaction buffer provided in the kit. Results were expressed as mean TG (mg/g tissue) �� SEM, n = 6 per diet group.

RNA isolation To detect early changes in gene expression, total RNA was prepared from the liver and visceral adipose tissue taken from 16-week-old mice Brefeldin_A (n = 4) in the four different diet groups using the Qiagen RNeasy kit according to the manufacturer’s instructions and stored at ?80oC. RNA quality was verified using a 2100 Bioanalyzer instrument and an RNA 6000 Nano LabChip assay (Agilent Technologies, Santa Clara, CA). RNA concentrations were determined by absorption at 260-nm wavelength with an ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE).

05), indicating substantial hepatoprotection by this procedure (T

05), indicating substantial hepatoprotection by this procedure (Table (Table4).4). In the further course, postischemic elevated ALT levels in both groups returned to normal within 7 d of hepatectomy. Serum bilirubin levels were determined as a parameter of hepatocellular function, but did not show notably different Ponatinib IC50 values at any time during the postoperative course (days 1-7, Table Table44). Table 4 Outcome data of patients undergoing liver resection with PM (A) or with IP + PM (B) Intraoperative parameters and postoperative course Blood loss as well as the need for autologous transfusion were significantly lower in the IP-treated group with 17% of patients receiving blood transfusion vs 48% in the control group (P < 0.05, Table Table4).4).

The postoperative course was uneventful in 24/30 (80%) patients in group B but only in 17/31 (53%) patients in group A (P < 0.05). Liver dysfunction, as previously defined, occurred in 2 patients of group A, but only in one patient of the IP-treated group (Table (Table4).4). Biliary leakage ceased spontaneously in 4 of the 6 patients (67%) of controls, but the other two patients required re-operation and bilioenteric anastomosis. In the study group, 2 patients had transient bile secretion and one patient of this group needed re-operation (bilioenteric anastomosis) (Table (Table44). Blood supply to the liver and hepatocellular injury With regard to earlier work, demonstrating a strong correlation between microcirculatory failure and postischemic enzyme release[22,26], it was of particular interest to determine whether there were changes in macrohemodynamic parameters, i.

e. the PM and IP may have an impact on parenchymal cell damage. Firstly, we analyzed the correlation between PV flow and ALT levels on day 1. Interestingly, by applying the Pearson Product Moment Correlation we did not find a significant association between the amount of the hepatocellular injury and quality of PV perfusion, either in controls (r = -0.38, P = 0.3) or in IP-treated patients (r = -0.41, P = 0.2). In contrast, when the HA flow of patients with PM (controls) and the corresponding ALT values on day 1 were analyzed, we found a weak, but significant inverse correlation, indicating a substantial influence of the macrocirculation at reperfusion on postischemic liver injury (r = -0.62, P = 0.042, Figure Figure2A).2A).

This correlation was even more evident, when patients underwent IP prior to PM as shown in Figure Figure2B2B (r = -0.73, P = 0.024), suggesting the HA perfusion was more susceptible to the procedure of IP in warm liver I/R. Figure 2 The Pearson product moment correlation between Cilengitide HA flow and alanine aminotransferase (ALT) levels. On day 1, there is an inverse correlation (P < 0.05) in the control group (A) undergoing PM (r = -0.62). In patients undergoing IP prior to PM (B), …

To colabel adipocytes with Bodipy-C12 and NBD-2-deoxyglucose (Inv

To colabel adipocytes with Bodipy-C12 and NBD-2-deoxyglucose (Invitrogen), explants were incubated for 1 h in glucose-free DMEM (Invitrogen) containing 10 nM insulin, washed twice with PBS, and then incubated for SKI-606 additional 10 min with 200 ��M NBD-2-deoxyglucose in 200 ��l of PBS at 37��C. Tissue was washed with PBS and overlaid with 200 ��l of M199 medium, and NBD fluorescence was collected as described below. Following NBD imaging, 200 ��l of prewarmed QBT Fatty Acid Uptake Kit (Molecular Devices, Sunnyvale, CA) was carefully added to the same well. Green fluorescent images were collected over 10 min of incubation. Confocal microscopy. Image recording was conducted using an inverted Leica SP5 AOBS spectral confocal system equipped with a motorized, temperature-controlled stage and HC PL FLUOTAR 10.

0 �� 0.30 and ��20 PL APO NA 0.70 dry objectives. Bodipy-C12 (excitation peak, 488 nm) was excited with an Argon laser, and images were recorded at emission bandwidth of 500�C550 nm. For QBT/NBD-2-deoxyglucose double-labeling experiments, NBD-2-deoxyglucose-labeled tissue was illuminated with an excitation wavelength of 488 nm (16% power), and fluorescence was collected at emission bandwidth of 498�C606 nm. Tissue was labeled with QBT (green Bodipy-C12) and illuminated with excitation wavelength of 488 nm (6% power), and fluorescence was collected at emission bandwidth of 500�C524 nm. Because NBD fluorescence appears weak compared with Bodipy fluorescence, it is possible to perform sequential double-labeling experiments by lowering excitation power and narrowing the emission bandwidth of the second (Bodipy) channel without a substantial bleed-through from the NBD channel.

WGA-Alexa fluor 633 was excited at 633 nm. In Bodipy/ethidium homodimer colabeling experiments, Bodipy was excited at 488 nm and fluorescence collected at 500�C550 nm, and ethidium was excited at 561 nm and fluorescence collected at 570�C650 nm. Image processing and analysis. The image analysis algorithm is illustrated in Supplemental Fig. S4 (Supplemental Material for this article can be found on the AJP-Endocrinology and Metabolism web site). Typically, 20�C30 z-sections, 5 ��m apart, were collected at 400 Hz, resulting in individual image dimensions of 1,550 �� 1,550 ��m. For high-resolution imaging, images were collected at 1-��m intervals.

Leica ��lif�� stacks of images were opened with the LOCI plug-in data browser and analyzed in Image J as follows. The stack of images was projected onto a single flat image (z-project, sum slices) containing integrated pixel intensities of the z-stack Entinostat (Supplemental Fig. S4A). The regions of interest (ROI), corresponding to cell boundaries, were drawn manually (ROI manager). Cells with poorly defined boundaries were excluded from analysis (Supplemental Fig. S1A).

This is coherent with the increased UPR activation observed in th

This is coherent with the increased UPR activation observed in the colonic tissue of active IBD patients, sellckchem whereas no increase was seen in the ileal tissue of active CD patients. In the ileum, ER stress is probably dictated by other local factors. The ileum contains a high number of Paneth cells, has an increased number of mucosa-associated E. coli and has a higher metabolic activity compared to the colon. This might contribute to a constitutive triggering of the UPR in the ileal mucosa, which is critical in maintaining homeostasis. The fact that inflammation does not further increase UPR in ileal samples either reflects that the higher basal levels observed can buffer some perturbations or reflect that the ileum is less sensitive to perturbations through inflammation.

This leads us to consider that the colonic mucosa is subject to a lower ER stress, with a significant increase in inflammatory conditions: from low basal levels of UPR, any induction is more uniform and more noticeable in this tissue. In order to determine whether the ileum could still respond to ER stress, paired colonic and ileal samples of five healthy controls were stimulated with tunicamycin, a well-known ER stress inducer [37], [38]. Both colonic and ileal samples revealed higher HSPA5 transcript levels in the tunicamycin stimulated samples. In addition, a higher induction was observed in the ileal samples. This would argue that the ileum rather lives on a higher basal ER stress, but can still induce strongly the gene response.

It also highlights that if the inflammation of the tissue did not significantly increase the UPR, it is not because the tissue is unable to do so. Indeed if the UPR is more activated in basal state, removal of a protective arm (XBP1) would prevent proper re-establishment of homeostasis and could logically result in imbalance. This would be coherent with both our findings and the ones of Kaser [27]. Moreover, in the study of Kaser, XBP1 was deleted exclusively in epithelial cells, pointing toward a defect in epithelial cells in IBD pathogenesis. In our study, we observed that HSPA5 located mainly in intestinal epithelial secretory cells (enteroendocrine cells, paneth cells and goblet cells) which produce large amounts of proteins involved in mucosal defense.

Regarding the activation of the PERK branch, transcript and protein levels of GADD34 in colonic IBD patients were similar to healthy controls, Carfilzomib which would argue against an activation of the PERK pathway. In contrast, western blot analysis showed an increased concentration of pEIF2A in colonic inflammation. If pEIF2A would result from PERK activation, we would expect an induction of GADD34, which is the co-factor of protein phosphatase 1 in the dephosphorylation of pEIF2A [32]. This would represent the canonical PERK pathway, where GADD34 promotes the return to homeostasis of the ER.


Enzastaurin LY317615 1% Triton-X. Cells were then washed three times with PBS and incubated with an appropriate fluorescein isothiocyanate-labeled secondary antibody (Jackson ImmunoResearch). Specimens were analyzed on an Olympus fluorescence microscope. For teratomas from ES cells, ES-Hepa hybrid cells and Hepa1�C6 tumors, ~1 �� 106 cells of each clone, were subcutaneously injected into the inguinal region of immunodeficient nude mice. The mice were monitored for tumor growth for 6 weeks. Tumor volume (V) was calculated in all experiments according to V = ab2/2, where a and b designate the long and short diameters of the tumor, respectively. At 4 weeks, teratomas or tumors were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.

In Vitro Differentiation For embryonic body formation, ES and ES-Hepa hybrid cells were harvested by treatment with 0.25% trypsin. The cells were cultured in bacterial-grade Petri dishes in Dulbecco’s modified Eagle’s medium/F-12 containing 20% knock-out serum replacement (Invitrogen), 2 mm l-glutamine, 1 �� 10?4 nonessential amino acids, 1 �� 10?4 M2-mercaptoethanol (Invitrogen), and 1% penicillin/streptomycin. The medium was changed every other day. Embryonic bodies were harvested at days 3, 5, 7, and 9 for gene expression analysis. For induction of ES and ES-Hepa hybrid cells to differentiate by monolayer culture, ES and ES-Hepa hybrid cells were dispersed into a single cell suspension with 0.25% trypsin, and plated the cells on the 0.2% gelatin-coated cell culture dish.

The cells were differentiated in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen) containing 15% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin/glutamine and changed the medium every 1 or 2 days. RNA Extraction, cDNA Synthesis, Quantitative PCR, and RT-PCR For real-time quantification of gene expression, cells were lysed with buffer RLT (Qiagen 79216), and Cilengitide RNA was extracted with RNeasy microcolumns (RNeasy lysis buffer, Qiagen), according to the manufacturer’s instructions. Random hexamer-primed first-strand cDNA was prepared with the SuperScript III reverse transcriptase kit (Invitrogen, cat. no. 18080-051) according to the manufacturer’s instructions. The raw quantification data for the transcripts were normalized to the endogenous GAPDH gene (ABI PRISM 7700 Sequence Detection System User Bulletin 2, Applied Biosystems). PCR primers are listed as follows.

However, the genetic polymorphisms in coding region alter the fun

However, the genetic polymorphisms in coding region alter the function or structure of encoded proteins. Those alterations can directly influence the phenotype of diseases. Therefore we assumed that the genetic polymorphism at codon 10 may affect the production or activity of TGF-��1, because it presents within the coding region. The function of a signal sequence moreover is translocation of newly synthesized proteins across the membrane of the endoplasmic reticulum (17). It consists of three regions: a positively charged NH2-terminal region, a central hydrophobic core, and a polar COOH-terminal region (18). The amino acid coded by codon 10 of TGF-��1 gene is located in the central hydrophobic core and influences on the overall hydrophobicity of core sequence (4).

Therefore, the change of a neutral proline to hydrophobic leucine residue disrupts the structure of the region, alters transportation across endoplasmic reticulum (4), and may affect the production or activity of TGF-��1. The serum TGF-��1 levels were not measured in present study, because TGF-��1 is locally synthesized in the liver and may not reach systemic circulation for measuring. In addition, because platelet is the main source of serum TGF-��1, the serum levels of TGF-��1 do not always reflect its production in liver (5). Therefore, the analysis of TGF-��1 mRNA and protein expression in the liver are required for evaluating phenotypic differences according to the genetic polymorphisms. TGF-��1 was also associated with the development of HCC (19) and the L/L genotype at codon 10 in TGF-��1 was associated with the development of HCC (7, 10).

In the present study, HCC was present in 58% of patients of LC group, and the fact might influence on the results of this study. However, 70-90% of the patients with HBsAg positive HCC had already underlying cirrhosis (19). In addition, when cirrhotic patients without HCC were selected for analysis, the L/L genotype was still a significant risk factor for the development of cirrhosis. Liver fibrosis is a highly complex disease process in which multiple genes interact with viral and environmental risk factors. Among them, age is a major risk factor for the development of cirrhosis irrespective of any etiology. In order to exclude the influence of age factor, present study’s inclusion criteria restricted the age Drug_discovery of patients over 50 yr old. The reasons why present study chose 50 yr old for age were; first, cirrhosis was generally diagnosed around this age (20) and second, it is not common that chronic HBV carriers remain chronic hepatitis without cirrhosis over this age in Korean population (personal experience). The longer duration of HBV infection is also an important risk factor of cirrhosis.

This meta-analysis also showed that multiple-item measures of sub

This meta-analysis also showed that multiple-item measures of subjective norms were a stronger predictor of intentions than single-item measures, which may explain our results. Although this was also found in an earlier study (Bledsoe, 2006), it is unclear why Fluoro Sorafenib self-efficacy was not significantly associated with intention after controlling for the other predictors in the model. A meta-analysis showed that attitudes, subjective norms, self-efficacy, and intention explain 27% of the variance in behavior (Armitage & Conner, 2001). Our explained variance was similar for quit attempts (28%), but much higher for quit success (50%). This may be caused by the strong direct effect from self-efficacy on quit success, which is consistent with predictions of social cognitive models.

Consistent with earlier studies (Borland et al., 2010; Hyland et al., 2006; Zhou et al., 2009), intention to quit was a stronger predictor of quit attempts than of quit success. Earlier studies that examined the direct effects of smoke-free legislation on smoking behavior found positive effects in some jurisdictions and not in others (Bajoga et al., 2011; Callinan et al., 2010). Earlier ITC studies have also found inconsistent evidence of an effect of smoke-free legislation on smoking cessation (Cooper, Borland, Yong, & Hyland, 2010; Fong, Hyland, et al., 2006; Hyland, Hassan, et al., 2009; Nagelhout, De Vries et al., 2012). The current study sheds some light on psychosocial factors that mediate the effects of smoke-free legislation on smoking cessation, that is, if the legislation is not supported by smokers and smokers do not change their attitudes about quitting, no effects on cessation are to be expected.

However, behavioral factors like implementing voluntary smoking bans in homes and cars and complying with smoke-free legislation might also explain why smoke-free legislation stimulates smoking cessation in some individuals and not in others. Moreover, implementation characteristics of the smoke-free legislation may influence whether smoke-free legislation increases Cilengitide smoking cessation. It is, for example, reported that support for smoke-free legislation increases more after the implementation of comprehensive than after the implementation of partial smoke-free legislation (Mons et al., 2012; Thrasher, Swayampakala, et al., 2010). Comprehensive smoke-free legislation may therefore lead to more smoking cessation than partial legislation. Also, smoke-free legislation that is implemented with accompanying media attention may lead to more support and harm awareness (Thrasher et al., 2011; Villalobos et al., 2010).

Smoking restrictions in the home may also prevent

Smoking restrictions in the home may also prevent Vismodegib solubility relapse among former smokers (Shields, 2007). Restrictions on smoking in the home may also be related to better psychological health as smoking is related to psychological symptoms, notably depression and anxiety (Boden, Fergusson, & Horwood, 2010; Degenhardt & Hall, 2001). In addition, it is possible that living in a home with rules against smoking may heighten individuals�� awareness about their health and thus support the adoption of a healthy lifestyle that not only excludes smoking but also includes healthy nutrition, exercise, and sleeping habits. Less exposure to smoke and a healthier lifestyle, in turn, may contribute to greater psychological well-being (Hamer, Stamatakis, & Batty, 2010; Wainwright et al., 2007).

Thus, the benefits of introducing smoking restrictions in one’s home may extend to other areas of health. The main goal of this study was thus to empirically test the hypothesis that smoking restrictions in the home are related to engaging in a healthy lifestyle and, ultimately, to greater psychological well-being. Support for this hypothesis is provided by research, which suggests that people tend to make improvements in several health behaviors (i.e., eating nutritious food, exercising, reducing substance use) concurrently (Unger, 1996). Implementation of one health-related behavior (here, introducing a smoking ban) may facilitate the adoption of other health behaviors (e.g., eating better food, exercising). For example, one study of middle-aged women found that those who had quit smoking reported higher levels of exercise and taking in a healthier diet (Perkins et al.

, 1993). Another study, applying the transtheoretical model (Prochaska & DiClemente, 1992), found that participants in the advanced stages of smoking cessation demonstrated more healthful behaviors than those in earlier stages (Unger, 1996). According to this model, individuals�� willingness to change their health behaviors can be classified into five stages along a continuum (precontemplation, contemplation, preparation, action, Entinostat and maintenance). Unger’s study, which examined the relationship between stages of smoking cessation and alcohol use, exercise, and safe driving practices, found that smokers who were not even considering quitting (i.e., precontemplators) also scored lower on other health behaviors. Specifically, precontemplators reported taking in more drinks per occasion than all other groups, binge-drank more frequently than all other groups, and exercised less than actors and maintainers (Unger, 1996). These findings show that not being willing to give up cigarette use is related to other health-compromising behaviors.

Nuclei were counterstained with 4��, 6-diamidino-2-phenylindole (

Nuclei were counterstained with 4��, 6-diamidino-2-phenylindole (DAPI), and specimens were visualized using an Axioplan2 fluorescence microscope (Carl Zeiss MicroImaging, Thornwood, NY). In addition, adult C57BL/6 mice were euthanized according to IACUC-approved protocols and urinary bladders were isolated, fixed in neutral-buffered selleck formalin for 2 h, dehydrated in graded alcohols, paraffin-embedded and cut into 5 ��m thick sections. Sections were analyzed for urothelial and epithelial markers as described above. Flow cytometry Following 14 d of culture, RA-stimulated and spontaneously differentiating UP2-GFP ESCs were dissociated into a single cell suspension and quantified for GFP fluorescence on a FACScaliber flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Untransfected ESCs either maintained as controls or differentiated similarly were analyzed in parallel in order to control for background fluorescence. CellQuest 3.0 software (BD Biosciences) was used to acquire and analyze FACScan data. In some experiments, GFP+ populations were sorted and collected from RA-treated UP2-GFP ESCs utilizing a MoFlo high-speed sorter (Dako-Cytomation, Carpinteria, CA) and subjected to further analysis detailed below. Protein Analysis Whole cell lysates were retrieved in 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitors. Nuclear extracts were obtained according to methods reported in the literature [37]. Immunoblot analysis was performed as described previously [38].

Primary antibodies included: anti-p63 and GATA4/6 as described above. Electromobility shift analysis Electromobility shift analyses (EMSAs) for GATA-4 and GATA-6 binding to murine uroplakins 1B and 2 promoter sequences were carried out as using methods previously described [39]. Briefly, 3�C5 ��g of nuclear extract and 0.5 ng of 32P-labeled oligonucleotide probe were combined in a reaction mix (20 ��l reaction volume) containing 25 mM Tris-HCl, pH 8, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 8% glycerol, 2 ��g of BSA, and 0.5 ��g of poly(dI-dC). The reaction was incubated for 20 min at room temperature, and the DNA-protein complexes were resolved on 5% polyacrylamide gels in 0.5X Tris-borate-EDTA (TBE) buffer at 4��C. The gels were dried, and the complexes were visualized by autoradiography using a Typhoon Trio variable mode phosphorimager (GE Healthcare Biosciences, Piscataway, NJ).

For competition experiments, a 25-fold molar excess of the cold competitor oligonucleotide was added simultaneously with the probe. For supershift experiments, a GATA4 or GATA6 antibody (as described above) was preincubated on ice for 1 h with nuclear extract followed by addition of the other components for 20 min at Cilengitide room temperature. Species-matched IgG antibodies were used in parallel as additional controls.