Fig. 3

Hypoxia induces cell membranous interaction between Pyk2 and NOX5. a KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was examined using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. b KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Intracellular Ca²⁺ level was evaluated using Calcium detection assay kit. c KYSE30 and KYSE410 cells were pretreated with 10 μM Ca²⁺ chelator-BAPTA-AM for 30 min and then exposed to normoxic or hypoxic condition for 1 h. The Pyk2 (Tyr⁴⁰²) phosphorylation was assayed by Pyk2 activation ELISA assay. d, e KYSE30 control, NOX5, or Pyk2-ovexexpressing cells were pretreated with 10 μM Ca²⁺ chelator-BAPTA-AM for 30 min or control solvent (d), or transfected with control vector or Pyk2 Y402F plasmid (e), were cultured normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was evaluated using immunoblotting to assess the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. f The KYSE30 and KYSE410 control shRNA or NOX5 shRNA cells were exposed to hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H₂O₂ was examined using an Amplex red hydrogen peroxide assay kit. g KYSE30 control or NOX5-overexpressing cells pretreated with or without 10 μM Ca²⁺ chelator-BAPTA-AM, or transfected with control vector or Pyk2 Y402F plasmid, were exposed to normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H₂O₂ was assayed using an Amplex red hydrogen peroxide assay kit. ***P < 0.001; two-tailed unpaired Student’s t-test. Error bars represent mean ± SD of five independent experiments