There were micrograms of protein extract were separated by SDS-polyacrylamide gel and transferred to nitrocellulose membranes using a Trans-Blot semi-dry transfer cell. After blocking in 1% skimmed milk drying the membranes were incubated with rabbit primary Rantik Body ER, rabbit anti-mGluR1 and rabbit anti-pAkt, by incubation with rabbit anti-horseradish peroxidase-conjugated secondary Rantik incubated Body, followed . Protein loading was with the fight against the act in select experiments, the same membranes then blotted with actin to k Mpfen again. Specific bands were visualized by verst Markets chemiluminescence using the Immobilon detection system detects. Follow the Rainbow field markings are used to determine the size E sch COLUMNS of the band Were. Densitometric analysis of Bandenintensit t was carried out with the aid of ImageJ software. Co-Immunpr Zipitation. Neurons were harvested in assay buffer Radioimmunpr Zipitation, and the protein concentration was determined by the Bradford method for co-Immunpr Zipitation, 500 g of protein in a final volume were determined from 500 l incubated for 1 to 4 hours in a shaker with 25 l of rabbit serum as nonspecific to reduce bond. Then, 20 l of the G protein PLUSAgarose were for 30 min at 4 to endogenous antibody To remove body. The samples were centrifuged and the whichever type Walls have been retained. Rabbit anti ER or rabbit anti mGluR1 was to whichever type Ligand was added and the mixture was placed in a rotary shaker at 4 to 19:00. Of the antibody Body-protein complex was adsorbed with 20 l of protein G PLUS agarose in a rotary shaker at 4 for 10 h and then washed five times with an L Solution, the PBS and 1% Tween 20. The samples were transmitted using the electrophoresis, SDS-polyacrylamide gel with 4-15% gradient gels and transferred to nitrocellulose membranes. After blocking in a PBS-L Solution with 2% skim milk and 0.1% Tween 20, membranes with primary Rem Antique Body rabbit anti bcr-abl incubated mGluR1 or rabbit anti-ER, followed by incubation with horseradish peroxidase conjugated anti-rabbit -Antique secondary body r. Detection of specific bands was performed with Immobilon detection system. Immunostaining staining.
The cells were fixed in 4% paraformaldehyde, with 0.1% Triton X-100 and saturated Saturated with 3% BSA. MGluR1 and rabbit anti mouse anti overnight at 4 ER, mouse anti-GFAP and mouse anti-MAP2 for 2 h at room temperature: The cells were then incubated with primary antibody Ren body incubated as follows. For fluorescent immunodetection, the following fluorochrome-conjugated antibody used body: Alexa Fluor 488 and mouse anti-rabbit-Texas Red. Studies in heterologous expression systems. HEK293 cells were grown in DMEM, erg complements With 10% FCS and antibiotics. Cells were transfected into bo her 10 mm with 10 l of Lipofectamine 2000 in Optimem medium and 18 g of the total cDNA as follows: 7.5 g mGlu1 receptor cDNA, 7.5 g of ER cDNA, and 3 g Tr ger of excitatory amino acids 1 cDNA. The transfections were carried out for 4 h, then the cells were plated in the culture medium in six well plates, which are previously coated with 0.01% poly-L lysine With the application of this procedure about 80 to 85% of HEK293 cells immunopositive cotransfected green fluorescent protein. The experiments were performed 72 h after transfection and serum starvation w Carried out during 16-18 h. Measuring the hydrolysis of polyphosphoinositide in cultured neurons.