Anti 16e7

HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37 °C with 5% CO₂. The cells were transfected with the various expression vectors using an established calcium phosphate method^{35 (link)}. To measure the half-life of Flag-tagged Rb mutants, transfected cells were metabolically labeled with [³⁵S] methionine/cysteine for 20 h, washed twice with phosphate-buffered saline (PBS), and chased for the indicated times in regular medium. Cells were harvested, washed with PBS, and lysed with buffer A (20 mM Tris-HCl [pH 7.4], 0.2% [v/v] NP-40, 150 mM NaCl, and 1 mM DTT) containing protease inhibitor cocktail set V (Millipore #539137). The extract was clarified by centrifugation at 20,000 × g for 10 min at 4 °C. PD150606 (Sigma #D5946), MLN4924 (Sigma #505477), carfilzomib (UBPBio #F1300), bortezomib (LC Laboratories #B-1408), MG132 (Enzo #BML-PI102), CIP (New England Biolabs, #M0290), anti-Flag (1:1,000; Sigma #F1804), anti-actin (1:1,000; Sigma #A2066), anti-16E7 (1:1,000; Santa Cruz #sc-51951), and InstantBlue (Expedeon #ISB1L) were purchased from the indicated manufactures. Quantification of bands was performed using ImageJ (National Institutes of Health, version 1.51).

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)].