Western Blotting

Cells were lysed using ice-cold RIPA buffer (Sigma) supplemented with phosphatase inhibitor cocktail (PhosSTOP, Roche) and protease inhibitor cocktail (cOmplete ULTRA Tablets, Roche). Insoluble material was removed by centrifugation at 12,000 rpm, 20 min at 4°C. Whole cell lysates were separated on 10 or 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred by electroblotting onto nitrocellulose membranes (Amersham Protran, GE Healthcare). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween20 (TBS-T) and incubated for 1 h. Western blots were performed using rabbit anti OTUD6B antibody (Abcam, #ab127714, 1:3,000), rabbit anti Cyclin D1 antibody (Cell signaling, #2978, 1:1,000), rabbit anti Cyclin D2 antibody (Cell signaling, #3741, 1:1,000), rabbit anti c-MYC antibody (Cell signaling, #13987, 1:1,000), rabbit anti Caspase 3 antibody (Cell signaling, #9662, 1:1,000) which detects both cleaved and uncleaved forms of caspase 3, rabbit anti E2F1 antibody (Abcam, #ab179445, 1:1,000), and rabbit anti GAPDH antibody (Cell signaling, #2118, 1:10,000). Bands were detected using goat anti rabbit secondary antibodies conjugated to horseradish peroxidase (Abcam, #ab6721, 1:5000). Cyclin D1, Cyclin D2, c-MYC, E2F1 expression was normalized to the expression of GAPDH, and cleaved caspase 3 expression was normalized to uncleaved caspase 3 expression. Calculation of the relative expression of protein was performed using ImageJ software.