Fig. 5
From: PRKCSH contributes to tumorigenesis by selective boosting of IRE1 signaling pathway
Internal E/P domain of PRKCSH is required for its interaction with IRE1α and boosting IRE1α activation under ER stress. a Immunoblot analysis of association between endogenous PRKCSH and IRE1α using immunoprecipitates from L02 cells treated with 10 μg/mL TM (left) or glucose-free medium (middle) for the indicated time, and from Huh-7 cells treated with 10 μg/mL TM (right) for the indicated time. b Immunoblot analysis of complex formation between endogenous IRE1α and Flag-tagged PRKCSH immunoprecipitated from transfected L02 cells treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-IRE1α antibody and normal rabbit IgG as a control antibody. c Immunoblot analysis of complex formation between endogenous PRKCSH and Flag-tagged IRE1α. Immunoprecipitates were prepared from transfected L02 cells treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-PRKCSH antibody and normal rabbit IgG. d Schematic diagram of the functional domains of PRKCSH and its recombinant variants (left part). Immunoblot analysis of in vitro complex formation between IRE1α and GST-tagged PRKCSH protein (right part). e, f Schematic presentation of wild-type and deletion mutants of PRKCSH (upper part). Immunoblot analysis of complex formation between endogenous IRE1α and Flag-tagged mutant PRKCSHs (lower part). Immunoprecipitates were prepared from transfected L02 cells treated with 10 μg/mL TM for 1 h. Immunocytochemical analysis of L02 cells transfected with Flag-tagged ΔG2B and ΔS/G2B mutant PRKCSH (f, middle). Calnexin was used as an ER marker. Scale bars represent 5 µm. g Immunoblot analysis of association between endogenous PRKCSH and GIIα. Immunoprecipitates were prepared from L02 (top) or Huh-7 cells (bottom) treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-GIIα antibody and normal rabbit IgG. h Model of PRKCSH complex formation. Under resting conditions, PRKCSH associates with the GIIα subunit via the G2B domain; this domain inhibits PRKCSH interaction with IRE1α. Under ER stress, PRKCSH dissociates from GIIα and then binds to IRE1α via the E/P domain
