Anti ubiquitin antibody

Total proteins were extracted for coimmunoprecipitation (co‐IP) assay. In brief, 400–500 μg protein was incubated with 2 μL anti‐14‐3‐3ζ at 4°C overnight, and then with 40–60 μL Protein A+G Agarose for 2–3 h. This mixture was centrifuged, washed with PBS, and then resuspended in 40–60 μL loading buffer (2×). After boiling for 5 min, 20–40 μL sample was subjected to Western blotting assay. Equal volume of each protein sample was separated on a 10% SDS‐PAGE and transferred onto PVDF sheets. The PVDF membranes containing target protein were blocked with skim milk, and then incubated with anti‐HBx (1:1000; Abcam), antiflag (1:500; CW‐Biotech, Beijing, China), or anti‐Ubiquitin antibody (1: 500; ProteinTech, Rocky Hill, NJ) at 4°C overnight, and then with proper secondary antibody (1: 5000, Beyotime) at 37°C for 45 min. After washing using TBST, the protein blots were developed by using an ECL reagent, and the densities were analyzed by Gel‐Pro Analyzer software. For Western blotting, total protein samples were used in the assay, with β‐actin as an endogenous reference. The primary anti‐Akt^{phospho S473} (1:1000) and anti‐Akt (1:5000) antibodies were purchased from Abcam.