Superlumia ecl plus hrp substrate kit

Treated samples were denatured and separated by SDS-polyacrylamide gel electrophoresis (Zhonghuihecai, Xian, China, PE008) and then transferred to PVDF membrane (Roche, Basel, Switzerland, 3010040001). The membrane was incubated with TBST containing 5% skimmed milk for 1 h at room temperature and reacted with the His monoclonal antibody (Abbkine, Wuhan, China, A02050) for 1 h. The membrane was incubated HRP-labeled secondary antibody. The signal was detected using superlumia ECL plus HRP substrate kit (Abbkine, Wuhan, China, K22030) according to manufacturer’s instruction.

The protein of RMICs was extracted using an ice‐cold lysis buffer containing phosphatase inhibitor and phenylmethanesulfonyl fluoride. The lysate was homogenized by a sonic oscillator then centrifuged at 12,000 rpm for 10 min at 4°C. Protein concentrations were determined using the Pierce TMBCA Protein Assay Kit. Equal amounts of sample protein (30–60 μg) mixed with 6 × loading buffer were separated by 8%–12% SDS/PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with 10% skim milk at room temperature for 1 h for blocking nonspecific binding sites and was then incubated with selected primary antibodies at 4°C overnight. The primary antibodies used in this study were anti‐FXR (1:500), anti‐TonEBP (1:1000), anti‐COX‐2 (1:1000), anti‐β‐actin (1:1000), anti‐pro‐caspase3 (1:1000) and anti‐cleaved caspase‐3 (1:1000). After being washed for 5 min with TBST buffer for five times, the membrane was incubated at room temperature with 1:4000 HRP‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h. The membrane was washed and detected using the SuperLumia ECL Plus HRP Substrate kit (K22030, Abbkine) and the images were collected with Tanon‐5200 (Tanon, Shanghai, China).

The heart tissue was homogenized in RIPA lysis buffer (Cwbiotech) and supplemented with 1 mM PMSF (Sigma) and 1× protease/phosphatase Inhibitor Cocktail (Cwbiotech). After quantification, each sample was denatured in SDS–polyacrylamide gel electrophoresis (PAGE) Loading Buffer (Cwbiotech) and boiled for 5 min. A total of 50 μg of protein was loaded in 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore). After blocking in TBST solution with 5% non-fat milk for 2 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies: anti-UCP3 (Zen-bio, 516996, 1:1,000) and anti-GAPDH (Abways, AB0036, 1:5,000). Membranes were then washed and incubated with species-specific HRP-conjugated secondary antibodies (Abways, AB0101, 1:5,000) at 1:5,000 in TBST with 5% non-fat milk for 1 h at room temperature. The blot was visualized by adding Superlumia ECL Plus HRP substrate kit (Abbkine) and then captured and analyzed by UVP Bio Imaging Systems (Sagecreation).