Anti ddb1

Cell lysates were prepared using RIPA lysis buffer (Pierce) containing a proteinase inhibitor cocktail (Roche Diagnostics). A total of 30 μg protein extracts were quantified and then subjected to electrophoresis on a 12% SDS-PAGE gel. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences) and blocked in Tris-buffered saline (TBS) containing 2% bovine serum albumin (BSA). The specific antibodies used including anti-FLAG (Cell Signaling Technology), anti-USP15 (Santa Cruz), anti-K48-linkage Specific Polyubiquitin (Cell Signaling Technology), anti-ubiquitin (Cell Signaling Technology), anti-DDB1(Cell Signaling Technology), anti-USP5 (Santa Cruz), and anti-β-actin(Sigma Aldrich). Proteins were detected by addition of alkaline phosphatase (AP)-conjugated secondary antibody. Visualization of the immunoreactive proteins was performed by addition of CDP STAR reagents (Roche). Densitometry analysis of band signals was carried out using Image J software and the levels of HBx protein in cells transfected with pHBx-FLAG + pUSP15-myc were expressed as the relative intensity (RI) to that in the empty vector control pUSP5-myc or pHBx-FLAG after normalization to β-actin.