FIGURE 6.

STRAP inhibits ASK1 downstream signaling. HEK293 cells transiently transfected with an empty vector (Vector), wild-type STRAP (WT), or mutant forms of STRAP (C152S/C270S and T175A/S179A) (A); parental HCT116 cells (B and C); and HCT116 cells stably overexpressing STRAP (STRAP(OE)) or HCT116 cells stably expressing STRAP-specific shRNA (STRAP(KD)) (B and C) were incubated with or without 2 mm H₂O₂ for 30 min. The endogenous activities of ASK1, MKK3, or p38 were determined by in vitro kinase assays or immunoblot analysis (WB) using the immunoprecipitates (IP) indicated or total cell lysates. The presence of equal amounts of each substrate in these assays was confirmed by immunoblotting with an anti-GST antibody. Overexpression or knockdown level of endogenous STRAP was determined by anti-STRAP immunoblotting. The relative level of kinase activity was quantified by densitometric analyses. The -fold increase relative to untreated samples in control HEK293 cells containing an empty vector alone or parental HCT116 cells was calculated.