Anti psma4

Frozen liver tissues were lysed in RIPA buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Servicebio, Wuhan, China) and 1 mM PMSF. Protein concentrations were determined using BCA Protein Assay Kit (Servicebio, Wuhan, China) by a microplate reader (PERL ONG, Beijing, China). Twenty micrograms of protein per lane were loaded onto an 8–12% polyacrylamide Gel and then transferred to PVDF membranes using an electrophoresis chamber (Millipore, Shanghai, China) for 1.5 h. Membranes were blocked in 5% non-fat milk Tris-buffered saline (TBS) followed by overnight incubation in primary antibody at 4°C. Primary antibodies used are as follows: anti-PSMB3 (#K009840P, Solarbio, Beijing, China), anti-PSMB6 (#K003745P, Solarbio, Beijing, China), anti-PSMA4 (#K003378P, Solarbio, Beijing, China) and anti-ATCB (#YT0099, Immunoway, TX, USA). Following primary antibody incubation, blots were washed four times for 10 min each time with TBS and 0.1% Tween 20, and incubated with an anti-rabbit IgG HRP-conjugated secondary antibody (#AS014, ABclonal, Wuhan, China). Blots were stripped with Enhanced Chemiluminescent Liquid (ECL, NCM, Suzhou, China) and re-probed with anti-ATCB for normalization. Blot imaging was performed on a chemiluminescence analyzer (Tanon-5200 Multi, Shanghai, China).