CAL27 and CCL138 cells were purchased from the American Type Culture Collection , and maintained in DMEM or EMEM and 10% FBS, penicillin and streptomycin, and hydrocortisone at 37”C with 5% CO2. HN5 cells were obtained from Ludwig Institute of Cancer Research FK-506 , and maintained in DMEM with 10% FBS, penicillin, and streptomycin, and hydrocortisone at 37”C with 5% CO2. For in vitro experiments, cell lines were grown to 70% to 80% confluence and then maintained for a further 48 hours in serumfree conditions before treatment. Serum starvation was undertaken to maximally synchronize cell cycle stage. Serum starved cells were treated with enzastaurin or rapamycin at the indicated concentrations for 30 minutes in serum containing medium.
Cells were then harvested and lysed, and proteins were subjected Tacrolimus clinical trial to protein immunoblotting analysis. Western Blot Analysis. Culture cells were rinsed twice in PBS, lysed in RIPA buffer with freshly added Phosphatase Inhibitor Cocktail Set II and Protease Inhibitor Cocktail Set III , scraped, and immediately transferred to microcentrifuge tubes. Protein yield was quantified with the Coomassie Protein Assay Kit . Samples containing equal amounts of total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked for 1 hour in Odyssey blocking buffer , and then incubated overnight at 4”C with the primary antibody diluted in Odyssey blocking buffer. The membranes were then washed thrice in PBS with 0.05% Tween 20.
Primary antibody was detected with the fluorescently labeled secondary antibody in Odyssey blocking buffer. Visualization and quantification were performed with Odyssey Infrared Imaging System . For total protein extraction from xenograft, mouse tissues frozen in liquid nitrogen after excision were homogenized in radioimmunoprecipitation assay buffer, Tacrolimus structure incubated for 15 minutes on ice, and spun in a centrifuge at 16,100g for 15 minutes. Western blot analysis was subsequently performed as described above. Integrated intensities of proteins generated by Odyssey Infrared system were normalized to the values obtained with a tubulin . The data obtained were then normalized by comparing with the data obtained with a reference standard sample, which was loaded into each group . Microwesterns.
Extracted mouse tissue was cooled in liquid nitrogen and pulverized to a fine powder with a mortar and pestle. Approximately 1 mg of lysate was added to 1 mL of Microwestern Tacrolimus solubility lysis buffer.10 The lysate was homogenized by boiling for 10 minutes and sonicating for 10 minutes wealth inequalities in a Diagenode Biorupter on high power, alternating 30 seconds on and 30 seconds off. This was followed by shearing with a 25 gauge needle 5 times. The lysate was concentrated fivefold in a 500 lL centricon The concentrated lysate was aliquoted and stored at 80”C until printing. Primary antibodies were incubated overnight at room temperature diluted in 100% Blocking Buffer . The blots were washed 4 times for 5 minutes in Trisbuffered saline solution with 0.1% tween, and with fluorescently conjugated secondary antibody for 1 hour in 20% Li cor buffer and 80% TBS without tween. The blots were washed three times in Tris buffered saline solution.