Fig. 4

Pyk2 recruits c-Abl to enhance NOX5 activity in Pyk2/NOX5 complex. a KYSE30 or KYSE410 cells were transfected with vector, Flag-Pyk2 wild type (wt), or Flag- Pyk2 Y881F mutant plasmid and then cultured under normoxia or hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with c-Abl antibody. Immunocomplexes were then immunoblotted using NOX5 and c-Abl antibodies (a). Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were measured using a modified OxyBlot protein detection kit (a). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) (a) in cell membrane lysis. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. b–d HeLa cells were co-transfected HA-c-Abl wt or kinase-dead (KD) K290R mutant with Flag-tagged NOX5 wt, Y476/478F, Y487F, or Y519F plasmid, respectively. Cell lysates were immunoprecipitated with the antibody against Flag. Cell membrane lysates were then immunoblotted using antibodies against flag and phosphotyrosine. Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were assayed using a modified OxyBlot protein detection kit (b). The transfection efficacy was measured using immunoblotting (c). The Pyk2 complex-derived H₂O₂ was tested using an Amplex red hydrogen peroxide assay kit (d). e, f The indicated ESCC cells were transfected with control vector, Flag-NOX5 wt, or Flag-NOX5 Y476/478F (mutant) plasmid, respectively, and then cultured under normoxic or hypoxic condition for 1 h. Oxidized Src levels were measured using a modified OxyBlot protein detection kit (e). The phosphorylation of Src Tyr⁴¹⁹ in Pyk2 complex was immunoprecipitated with the antibody against Pyk2 and then immunoblotted using antibodies against Pyk2 and pSrc (Tyr⁴¹⁹) (e). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) (e) in cell membrane lysis. The Pyk2 complex-produced H₂O₂ was assayed using an Amplex red hydrogen peroxide assay kit (f). ***P < 0.001; two-tailed unpaired Student’s t-test. Error bars represent mean ± SD of five independent experiments