Fig. 5
JNK1 associates with PRDX1 under redox (H2O2) stress and JNK inhibition reduces JNK signaling and characteristics found in CAFs. a-b 293 T cells were transfected with pcDNA3-FLAG-JNK1a1 (Addgene) and treated with increasing concentrations of H2O2 for 30 min. Before lysis, cells were washed with 20 mM N-ethylmaleimide (NEM) in PBS to block lysis-induced disulfide bond formation. FLAG-labeled proteins were immunoprecipitated and detected by immunoblot with FLAG, PRDX1, and PRDXSO2/3 antibodies. Note: JNK1 phosphorylation in the untreated sample may be enhanced by JNK1 over-expression. b Protein lysates from (a) were analyzed by immunoblotting with antibodies detecting JNK, PRDX1, and PRDXSO2/3. c Immortalized Prdx1−/− and Prdx1+/+ MFs were starved for 48 h before treated with 25 μM SP600125 for 1 h followed by 100 μM H2O2 treatment for 15 min. Proteins were analyzed by immunoblotting for phosphorylation of c-jun and JNK. d Immortalized Prdx1−/− and Prdx1+/+ MFs were starved for 48 h before treatment with 25 μM SP600125 for 1 h followed by 100 μM H2O2 treatment for 15 min and analyzed 48 h later for collagen, FAP, α-SMA vimentin, and caveolin 1, which was used as a loading control. e and f Immortalized Prdx1−/− and Prdx1+/+ MFs were starved for 48 h before treated with 25 μM SP600125 for 1 h followed by 100 μM H2O2 treatment for 15 min and analyzed for invasion by Transwell assays. Values indicate the mean ± SD for 3 repeats. Individual treatment samples were analyzed for statistical significance by Students t-test