Protein Expression Analysis in Keratinocytes

SDS-lysed keratinocyte cell lysates were equalized for protein concentration using BioRad reagents. Equal amounts of protein-normalized samples were loaded onto SDS-acrylamide gels, electrophoresed, and transferred onto PVDF membranes. Blots were blocked in 0.05% tween-20/5% Non-fat milk in Tris-buffered saline and probed with the indicated antibodies to GAPDH (#368 from Cell Signaling), Tubulin (#T9026 from BD Biosciences), anti-16E7 (a mix of both monoclonal antibody clones 8C9 and EDV7 from Santa Cruz Biotechnology), and antibodies from ThermoFischer to actin (MS-1295-P1) and p53 (MA1-19055). Anti-AU1 tag monoclonal antibody was a gift of Richard Schlegel (Georgetown University). Anti-16E6 MAb 6G6 was a kind gift of Johannes Schweitzer (Arbor Vita Corp.); lysates for 16E6 western blots were prepared from Igepal-lysed cells as described [45 (link)].