Horseradish peroxidase conjugated anti igg

Total lung lysates were prepared by homogenizing lung tissues in Laemmli buffer. Proteins were separated by SDS-PAGE and electroblotted onto Immobilon-P membranes. Western blotting analyses were carried out as previously described32 (link). The antibodies used in this study included: TGF-β1, TGF-β3 (Abcam), fibronectin (Sigma-Aldrich) and α-smooth muscle actin (SMA) (Millipore). Secondary antibody was horseradish peroxidase-conjugated anti-IgG (Santa Cruz Biotechnology). Signals were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce). The relative amount of proteins was quantified using gel analysis software UNSCAN-IT gel version 5.3 (Silk Scientific, Orem, UT), and normalized to β-actin internal loading control.

The treated cells in each group were washed twice with ice-cold PBS and then lysed in 0.5 ml of lysis buffer for 20 min at 4°C. Next, the lysates were centrifuged for 10 min at 12,000 g and 4°C, and the supernatants were collected. The protein concentration in each supernatant was determined using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Next, an equal amount of protein from each supernatant was separated by 10% SDS-PAGE, and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked with skim milk. The membranes were then incubated with primary antibodies against cleaved-caspase-3 (CST, Danvers, MA, U.S.A., 9654s), IL-2 (CST, D7A5), IL-6 (CST, D3K2N), P-p65 (CST, 93H1), p-65 (CST, D14E12), TNF-α (CST, 3707), FASLG (Abcam, Cambridge, MA, U.S.A., ab15285), and GAPDH (CST, 14C10) overnight at 4°C; After which, the membranes were incubated with horseradish peroxidase-conjugated anti-IgG (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) as the secondary antibody. The immunostained protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, U.S.A.).

A Western blot assay was performed as described^{28 (link)}. For Western blot analysis of whole-cell extracts, the following primary antibodies were used: TGF-β1 (1:5000; Cell Signaling Technology, Danvers, MA, USA), α-SMA (1:5000; Abcam,), and Col I (1:5000; Abcam). Smad2 (1:1,000 dilution, Protein-Tech, China), phospho-Smad2 (1:1,000 dilution, Protein-Tech, China), Smad3 antibody (1:1,000 dilution, Protein-Tech, China), phospho-Smad3 (1:1,000 dilution, Protein-Tech, China), p62 (1:1,000 dilution, ProteinTech, China), α-tubulin (1:1,000 dilution, Affinity, China). The proteins were separated using SDS-PAGE and transferred onto a membrane, followed by incubation with horseradish peroxidase-conjugated anti-IgG (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using an enhanced chemiluminescence kit (Tanon; Tanon Science & Technology, Shanghai, China). To ensure accurate quantification, the Results were normalized with GAPDH (1:2000; Abcam).