Kf005

Clinical LF specimens (non-LFH group:LFH group = 2:2) were snap-frozen in liquid nitrogen and stored at −80°C for Western blotting. Total protein from each LF specimen was extracted in RIPA lysis buffer (Santa Cruz) and quantified using the BCA assay (Pierce). After denaturation, proteins specimens were separated using gel electrophoresis on 8%–12% SDS-PAGE, and then were transferred onto PVDF membranes (Roche Applied Science, Indianapolis, IN, USA). Using 5% nonfat dry milk for 2 hours at room temperature, The membranes were blocked in 5% nonfat dry milk for 2 h and then incubated overnight at 4°C with the following primary antibodies: Beclin1 (1:500; AF5128, Affinity), P62 (1:500; AF5384, Affinity), FN1 (1:500; AF5335, Affinity), TGFβ1 (1:500; AF1027, Affinity), NGF (1:500; AF5172, Affinity), HMOX1 (1:500; AF5393, Affinity), CAT (1:500; DF7545, Affinity), SIRT1 (1:500; TU365233, Abmart), and GAPDH (1:5000; AP0063, Bioworld). After, the membranes were incubated with goat anti-rabbit IgG (H+L) HRP secondary antibody (1:5000; RM3002, Rayantibody) for 2 h at room temperature. The proteins bands were detected using an enhanced chemiluminescence kit (KF005, Affinity), and chemiluminescence signals were quantified with Image Lab statistical software (Bio-Rad, Hercules, CA, USA).

Cell lysates from the experimental and control groups were collected and prepared for western blot. Primary antibodies against CRLF1 (1:1000, ab211438, Abcam), collagen I(1:1000, AF7001, Affinity), collagen III (1:1000, GTX111643, Gene Tex), MMP2 (1:1000, AF5300, Affinity), a-SMA (1:1000, ab7817, Abcam) and GAPDH (1:5000, RM2002, Rayantibody) were incubated overnight at 4°C followed by incubation with the appropriate secondary antibodies: goat anti-rabbit IgG (H+L)-HRP (1:3000, RM3002, Rayantibody) and goat anti-mouse IgG (H+L)-HRP (1:3000, RM30011, Rayantibody). Proteins were detected using an enhanced chemiluminescence kit (KF005, Affinity).