Total protein samples were extracted from OS and OC tissues and from OS cells that were transfected with HDAC4-GFP or HDAC4 siRNA after transfection for 48 h by using cell lysis buffer (Thermo Scientific Pierce IP Lysis Buffer). The protein concentration was measured with a BCA Protein Assay Kit (Pierce). Fifty micrograms of protein per sample was used in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in Tris buffered saline with Tween 20 (TBST) at 37°C. After 1 h, the membranes were incubated with primary antibodies against HDAC4 (1:1,000, Abcam ab12172) and PCNA (1:500, Santa Cruz Biotechnology sc-25280) at 4°C overnight on a shaker. The next day, secondary antibodies were used to stain the membranes at 37°C for 1 h. The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000, Santa Cruz sc-2004) and anti-mouse IgG (1:10,000, Santa Cruz sc-2005). The detection was performed by using an enhanced chemiluminescence (ECL) kit.
The in vitro ubiquitination assays were performed as described above. When total protein samples were extracted from OS cells after transfection for 48 h, ubiquitinated PCNA was detected by SDS-PAGE and probed with primary antibodies (1:1,000, Cell Signaling Technology D5C7P).
The in vitro ubiquitination assays were performed as described above. When total protein samples were extracted from OS cells after transfection for 48 h, ubiquitinated PCNA was detected by SDS-PAGE and probed with primary antibodies (1:1,000, Cell Signaling Technology D5C7P).