Fig 2: Act1 (D10N) is a loss of function variant(a) Act1-/- MEFs transduced with vector, Act1 (WT), or Act1 (D10N) were subjected to immunoblot analysis for Act1 expression. (b) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times. Cell lysates were subjected to gel-shift assay for NF-?B activation. (c) HEK293 cells transfected with E-selectin–luciferase reporter (100ng) and the indicated amounts of human Act1 or human Act1 (D10N) DNA, followed by luciferase assay analysis of NF-?B activity. (d) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times, followed by immunoblot analysis. Arrow indicates Act1-modification. (e) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti-Act1, followed by immunoblot analysis for IL-17R, TRAF6, TRAF3, TRAF2, IKKi and Act1. Immunoprecipitated (IP) products are shown on left, whole cell lysates (WCL) are shown on right. (f) Act1-/- MEFs transduced with vector, Act1 (WT), Act1 (TB12), or Act1 (D10N) were left untreated or treated for 3 hours with TNF (10ng/ml), IL-17A (50ng/ml), IL-17F (50ng/ml), or TNF in combination with IL-17A, followed by RT-PCR analysis for Il6, Cxcl1 and Csf2 expression. The data are shown as fold induction over untreated (UT). *p < 0.05, **p < 0.01 and ***p < 0.005 (Student’s t-test). Data are representative of three independent experiments.

Fig 3: IL-17-induced Act1-Hsp90 interaction is TRAF6 independent(a) Schematics of TRAF-binding site mutant (TB12) of Act1. (b) Act1-/- MEFs transduced with Act1 (WT) or Act1 (TB12) were treated with IL-17 (50ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti-Act1, followed by immunoblot analysis for TRAF6, TRAF2, TRAF3, Hsp90, IKKi and Act1. (c) Lysates from WT or Traf6-/- MEFs treated with IL-17 (50ng/ml) for the indicated times were immunoprecipitated with anti-Act1, followed by immunoblot analysis for Hsp90 and Act1. (d) Act1-/- MEFs transduced with Act1 (WT), Act1 (D10N), or Act1 (TB12) were left untreated (0) or treated with varying concentrations of PU-H71 for 24 hours. The relative amount of total Act1 to actin was quantified by densitometry. The Act1 to actin ratio in untreated MEFs was defined as 1. (e) Act1-/- MEFs transduced as in (d) were treated for the indicated times with IL-17 (50ng/ml). Cell lysates were immunoprecipitated with anti-Act1, followed by immunoblot analysis for Hsp90, p23, HOP, Hsp70 and Act1. (f) Act1-/- MEFs transduced as in (d) were treated for the indicated times with PU-H71 (1µM). Cell lysates were immunoprecipitated with anti-Act1, followed by immunoblot analysis for Hsp90, p23, HOP, Hsp70 and Act1. Immunoprecipitated (IP) products are shown on left, whole cell lysates (WCL) are shown on right. Data are representative of three independent experiments.

Fig 4: Act1 is a client protein of HSP90(a) Lysates from MEFs treated with IL-17 (50ng/ml) for the indicated times were immunoprecipitated with anti-Act1 or IgG control, followed by immunoblot analysis for Hsp90, IKKi and Act1. Immunoprecipitated (IP) products are shown on left, whole cell lysates (WCL) are shown on right. (b) MEFs were treated with Hsp90 inhibitors 17-AAG (1µM) or PU-H71 (1µM) for the indicated times. Actin is used as loading control. The relative amount of total Act1 to actin was quantified by densitometry. The Act1 to actin ratio in untreated MEFs was defined as 1. (c) DMSO (control) or GA pretreated MEFs were incubated with 10 µg/ml cycloheximide (CHX) for the indicated times. The relative amount of total Act1 to actin was quantified by densitometry. The Act1 to actin ratio prior to CHX treatment was defined as 1. (d) MEFs were treated with DMSO, PU-H71 (0.5µM) alone, MG-132 (1µM) alone, or PU-H71 (0.5µM) plus MG-132 (1µM) for 12 hours. The relative amount of total Act1 to actin was quantified by densitometry. The Act1 to actin ratio in DMSO-treated MEFs was defined as 1. (e) Lysates from MEFs treated with PU-H71 (1µM) for the indicated times were immunoprecipitated with anti-Act1 or IgG control. The data are representative of three independent experiments.

Fig 5: IKKi expression in the hypertrophic heart.A,Western blot analysis of the cardiac IKKi protein in WT mice after aortic banding at the time points indicated (n = 6). B, RT-PCR analysis of cardiac IKKi mRNA levels in WT mice after aortic banding at the time points indicated (n = 6). *P<0.05 vs. sham.