Chemido xrs gel imager system

The liver tissues were homogenized and lysed in ice-cold lysis buffer supplemented with protease and phosphatase inhibitors (Topscience, Shanghai, China) and centrifuged at 12,000 g for 20 min at 4°C, and then the clarified supernatants were collected. Then proteins were quantified with the Pierce BCA Protein Assay Kit (Thermo, United States) assay according to the manufacturer’s instructions. The western blot was performed as previously described (Li et al., 2022a (link); Li et al., 2022b (link)). Briefly, after SDS-PAGE and transmembrane, the target proteins were accordingly probed with first antibodies against GAPDH (10494-1-AP, 1:1,000, Proteintech), phospho-Stat3 (9145, 1:1,000, CST), Stat3 (9139, 1:1,000, CST), phospho-p38 MAPK (4511, 1:1,000, CST), p38 MAPK (8690, 1:1,000, CST), phospho-NF-κB p65 (3033, 1:1,000, CST), NF-κB p65 (6956, 1:1,000, CST), phospho-IκBα (2859, 1:1,000, CST), and IκBα (4814, 1:1,000, CST), respectively. After incubation with the corresponding HRP-conjugated secondary antibody, the signal of the target protein was detected using a ChemiDo XRS gel imager system (Bio-Rad, United States) with Immobilon Western Chemiluminescent HRP Substrate (Millipore, United States) and was scanned by ImageJ software. The ratio of the target protein was normalized to the internal control protein GAPDH, and fold-change was calculated relative to the control group.

Western blot (WB) was performed as previously described [25 (link)]. Briefly, after the membrane was washed, the target proteins were accordingly probed with the antibody to HCV Core (1 : 2500) or NS3 (1 : 2500). As an internal control, the antibody to actin (1 : 4000) was used. After having been washed with TBST, the membrane was incubated with the goat anti-mouse or goat anti-rabbit secondary antibody, respectively. The proteins were detected using an Immobilon Western Chemiluminescent HRP Substrate (Millipore, Inc.) with ChemiDo XRS gel imager system (Bio-Rad, CA). The protein signal intensity was scanned with the Gelpro32 software, and a ratio of the interested protein to the internal control protein actin was calculated and normalized as 1.00 for the control group.

The Western blot analysis was performed as previously described.²¹ In brief, equal amounts of protein from cellular lysates were subjected to SDS‐PAGE and electroblotted onto transmembrane (Millipore). Proteins were probed with antibodies against HCV core, TGF‐β1, TGF‐β3, His tag, TβRI or SMAD2/3, respectively. β‐Actin antibody was used as an internal control. Proteins were detected by chemiluminescence with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized by ChemiDo XRS gel imager system (Bio‐Rad). Protein band intensity was analysed by Gelpro32 software. The ratio of interested protein to internal control protein β‐Actin was calculated and normalized as 1.00 for the control group.