Af5335

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).

Immunoblotting was done by a method as described previously [14 (link)]. Protein quantification was done by Bradford assay and equal quantity of protein was loaded onto each lane. The primary antibodies used in the study were mouse anti-α-SMA (ab7817, 1:1000, Abcam, Cambridge, MA, USA), anti-vimentin (D21H3, 1:1000, Cell Signalling Technology, Beverly, MA, USA) rabbit anti-fibronectin (AF5335, 1:1000, Affinity Biosciences, Brisbane, Queensland, Australia), mouse anti-fibulin-5 (ab66339, 1:800, Abcam, Cambridge, MA, USA), mouse anti-PAI-1 (ab125687, 1:1000, Abcam, Cambridge, MA, USA), rabbit anti-Fibrillin (AF0429, 1:1000, Affinity Biosciences, Brisbane, Queensland, Australia) and rabbit anti-pSMAD3(C25A9, 1:1000, Cell Signalling Technology, Beverly, MA, USA). Rabbit anti-GAPDH (1:10,000; Abcam, Cambridge, MA, USA) was used as the loading control.

Paraffin-embedded lung tissue was dewaxed with xylene, and the sections were heated in a microwave oven with antigen-fixing solution (0.01 M citrate buffer) for 20 min. After the sections were cooled to room temperature and blocked with an immunohistochemistry kit, the primary antibody was added and incubated at 4 °C overnight. The primary antibodies were as follows: mouse anti-α-SMA (1:200 dilution, Affinity, BF9212), mouse anti-fibronectin (1:200 dilution, Affinity, AF5335). After being washed with TBST three times, the tissue sections were incubated with the secondary antibody at room temperature for 1 h. Subsequently, the tissue sections were rehydrated through a series of ethanol solutions and were stained with hematoxylin to observe histological changes and target gene expression under an optical microscope.