Example 4
Cells were treated with experimental compound for 12 hrs then lysed in either NP-40 lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA) containing protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 17000×g for 12 minutes and the supernatant was collected for immunoblot analysis. Equal amounts of protein were separated by SDS-PAGE on pre-cast 2-20% polyacrylamide gradient gels (Biorad) and electroblotted onto Immobilin poly-vinyl difluoride (PVDF) membrane (EMD Millipore). Membranes were blocked in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk for 1.5 hours at room temperature, followed by overnight incubation with primary antibodies diluted in blocking buffer at 4° C. (anti-N17-S13pS16p 1:2500; anti-p53-S392p 1:1500; anti-GAPDH 1:7500). After three 15-minute TBS-T washes, membranes were incubated with HRP-conjugated secondary antibodies diluted in blocking buffer (1:50000) for 45 minutes at room temperature, and visualized using Immobilin enhanced chemiluminescence HRP substrate (EMD Millipore) on a MicroChemi system (DNR Bio-imaging Systems). Bands were quantified using NIH Image J software and normalized to GAPDH controls (