Pathscan acetylated p53 sandwich elisa kit

For protein analysis, fresh frozen brains were thawed to 20°C and the left hemisphere was dissected. Partial samples of somatosensory cortex, hippocampus, and the cerebellum were obtained using a beveled 16G needle as a hole punch. These specimens were immediately placed in 500 μL of Qiazol (Qiagen, Valencia, CA). Protein/DNA/RNA purification was done following the Trizol extraction method. Protein was reconstituted in 50 μL of 1% SDS with 1:100 protease inhibitor cocktail and quantified using the Micro BCA Protein Assay Kit (Pierce, Waltham, MA) and measured on a plate reader at 562nm. Total p53 was measured using 1 μg total protein with the MyBioSource ELISA kit San Diego, CA (Cat. # MBS076677). Acetylated p53 was measured using 1 μg total protein with the Cell Signaling Technology (Danvers, MA) PathScan Acetylated p53 sandwich ELISA kit (Cat. #7236). Analysis of the protein data was performed using a two-way ANOVA to test for significant main effects of ethanol Treatment and p53 Genotype, as well as their interaction.

Evaluation of p53 acetylation in SMCs was done by a PathScan Acetylated p53 Sandwich ELISA kit (Cell Signaling 7236C), following manufacturer instructions. Briefly, total cell lysates were obtained, sonicated and protein concentration measured. Then, 100 μl of each sample containing equal amount of protein was added to a microwell coated with a p53 mouse monoclonal antibody and incubated at 4 °C overnight. The microwells were extensively washed, and an acetylated-lysine rabbit monoclonal antibody added for 1 h at 37 °C. After washing, an anti-rabbit horseradish peroxidase-linked antibody was added for 0.5 h at 37 °C. After washing, 100 μl of TMB substrate was added for 10 min at 37 °C. Then, 100 μl of STOP solution was added and absorbance at 450 nm read in a Synergy 2 Microplate Reader (BioTek). The magnitude of the absorbance is proportional to the quantity of acetylated p53. Triplicates for every genotype and condition were used every time. When indicated, cells were treated with 10 μM RO5963 (Calbiochem 44153), 50 μM of ICG001 (SelleckBio S2662) or vehicle (DMSO); or transfected with CBP siRNA (Santa Cruz sc-29243) or control siRNA (Santa Cruz sc-44231) using X-tremGENE siRNA transfection reagent (Roche 04476093001).