Fig. 6: HOPS interferes with IκBα stability in the absence of direct binding.

A Expression of p-IκBα (Ser32) and total IκBα in RAW 264.7 cells transfected with or without myc-HOPS and treated with LPS for the indicated times. Anti-myc antibody was used as control of transfection. α-tubulin was used as loading control. Densitometric analysis for p-IκBα phosphorylation rate versus total IκBα expression levels is shown on the right. B Expression of IκBα in RAW 264.7 cells transfected with or without myc-HOPS and treated with CHX (100 μM) for the indicated times. α-tubulin was used as loading control. Densitometry for IκBα expression levels is shown on the right. C HEK 293T cells were cotransfected with myc-HOPS and Flag-p65. p65 was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated proteins were revealed by WB analysis with anti-IκBα anti-Flag antibodies. Total cell lysate input was revealed with anti-Flag, anti-IκBα and anti-myc antibodies. D HEK 293T cells were transfected with HA-(8X)ubiquitin and myc-HOPS or empty vector. 24 h post-transfection cells were exposed to MG132 (10 μM) for 6 h. 30 min before lysis, cells were treated with LPS (10 μg/ml) or left untreated. Anti-IκBα immunoprecipitations were immunoblotted with anti-HA to reveal the amount of ubiquitin-bound IκBα. IκBα and HOPS (anti-myc) were probed in total cell lysates as experimental input (E) HEK 293T cells were cotransfected with myc-HOPS and Flag-p65. HOPS was immunoprecipitated with anti-myc antibody, and coimmunoprecipitated proteins were revealed by WB analysis with anti-IκBα and anti-myc antibodies. Total cell lysate input was revealed with anti-Flag, anti-IκBα and anti-myc antibodies.