Anti α sma bm0002

Fresh mouse livers were ground into powder in liquid nitrogen, and moderate protein lysis solution (RIPA : PMSF = 100 : 1) was added. After incubation on ice for 30 min, tissue debris was removed by centrifugation (15 min, 4°C). Protein concentrations were assayed by using the Bradford assay (BIO-RAD). Total protein was resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (0.2 μm, Millipore). After blocking with 5% skimmed milk, membranes were probed with the appropriate antibody. Protein bands were detected with ECL reagents. The antibodies used were as follows: anti-GAPDH Ab (Cell Signaling Technology), anti-α-SMA (BM0002, BOSTER), anti-α-SMA (BM0002, BOSTER), and anti-tTG Ab (sc-20621, Santa Cruz Biotechnology).

To reveal NF-κB p65 and iNOS proteins in murine macrophages RAW 264.7 and Desmin and α-SMA in primary murine HSCs, double immunostaining was conducted. Paraformaldehyde-fixed cells were incubated with the primary antibody, followed by the secondary antibody, and then mounted with DAPI. The primary antibodies included anti-NF-κB p65 (1:200), anti-iNOS (1:200), anti-Desmin (16520-1-AP) (Proteintech, Wuhan, China) (1:200), anti-α-SMA (BM0002) (Boster, Wuhan, China) (1:200). The secondary antibodies included FITC-labelled goat anti-rabbit IgG (a0562) (H+L; Beyotime, Hainan, China) (1:200) and Cy3-labelled goat anti-mouse IgG (a0521) (H+L; Beyotime, Hainan, China) (1:200).

Treated cells were lysed for 30 min on ice in RIPA buffer containing a protease inhibitor cocktail (Meilun, Dalian, China). Equal protein loading was ensured by measuring the protein concentration in each sample using a BCA protein assay kit (Meilun, Dalian, China). Loading buffer was added to the protein, and the mixture was heated at 98°C for 10 min. Subsequently, the samples were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with protein-free rapid blocking buffer (Meilun, Dalian, China) for 10 minutes, and probed with the following primary antibodies: anti-α-SMA (BM0002, Boster, Wuhan, China), anti-CNN1 (abs171608, Absin, Shanghai, China), anti-MYH11 (K002095P, Solarbio, Beijing, China), anti-MMP2 (A00286-2, Boster, Wuhan, China), and anti-MMP9 antibodies (BM4089, Boster, Wuhan, China). After three washes, the membranes were incubated with HRP peroxidase-conjugated AffiniPure goat anti-rabbit IgG (BA1056, Boster, Wuhan, China) for 1 h. The signals were detected and visualised using a chemiluminescence imaging system (Millipore, Billerica, MA, USA). Subsequently, the protein levels were quantified and assessed using ImageJ software and subsequently normalised based on the corresponding β-actin levels.