Anti phospho erk1 2 thr202 tyr204

The lung tissues from the experimental rats and lung fibroblasts treated with Chol-HCQ were homogenized in RIPA lysis buffer (Beyotime Biotech, China) containing 1 mM phenylmethylsuphonyl fluoride. The lysates were then centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were collected and stored at −80 °C. A BCA protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine the protein concentrations. Equal amounts of protein were loaded and run on 10% SDS-PAGE gels, transferred onto Millipore PVDF membranes and blocked with 4% BSA. Then, the membranes were incubated with primary antibodies at 4 °C. The following primary antibodies were used: (1) anti-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (2) anti-phospho-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (3) anti-ERK1/2 (AbcamPLC, Boston, MA, USA), and (4) anti-phospho-ERK1/2 (Thr202/tyr204) (AbcamPLC, Boston, MA, USA). Antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody and the blots were developed with the ECL-Plus reagent (Millipore, MA, USA). The blots were tested for GAPDH (AbcamPLC, Boston, MA, USA) to confirm equal protein loading.