P smad

The cultured cells were lysed in RIPA buffer plus protease inhibitor (PMSF). Total cellular protein was collected by centrifugation, and the concentration determined by the BCA method. Proteins were first separated by SDS-PAGE and then transferred to PVDF membranes (0.45 µm, Millipore). The PVDF membranes were washed with TBST, blocked for 1 h with 5% skim milk powder dissolved in TBST, and incubated with primary antibodies against TGF-β (Calbiochem, MA, USA), Smad3 (Santa Cruz, CA, USA), p-Smad (Santa Cruz, CA, USA), Collagen I (Merck Millipore, MA, USA), C3a (Abcam, MA, USA), C3aR (GeneTex Inc., USA), IL-6 (LSBio, Seattle, WA, USA), p-IKBα (Abcam, MA, USA) and IKBα (Santa Cruz, CA, USA) at 4°C overnight with dilutions recommended by the manufacturer. β-actin was used as an internal reference. The PVDF membranes were washed with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at 37°C for 1 h. Protein bands were detected using chemiluminescence (ECL) reagent (Pierce, Thermo Scientific). The quantitative analysis of protein band density was performed on Quantity One (Bio-Rad).

HSC-T6 cells were seeded in six-well plates at a density of 2 × 10⁵ cell/well and incubated with 68 μM (IC₅₀) and 34 μM (half IC₅₀) of WM130 and 68 μM of M19 for 24 h; PBS was used as blank control. Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with 5% nonfat dry milk and incubated with the primary antibodies, including the rabbit antibodies to rat AKT, p-AKT, ERK, p-ERK, EGFR, p-EGFR, and p-Raf (Cell Signaling Technology, Danvers, MA), the mouse antibodies to rat TGF-β1, α-SMA (Abcam Inc. Cambridge, MA) and p-Smad (Santa Cruz Biotech Inc., CA), and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Kangchen Bio-tech, Shanghai, China), followed by incubation with the secondary antibodies, horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (Ig) G or sheep anti-rabbit IgG (Abcam Inc. Cambridge, MA). Blots were developed using an enhanced chemiluminescence detection kit (Beyotime Biotechnology, Shanghai, China). The agarose gel bands were scanned for gray density analysis relative to the loading control by ImageJ software.