Fig. 5: HOPS increases p65 phosphorylation.
From: HOPS/Tmub1 involvement in the NF-kB-mediated inflammatory response through the modulation of TRAF6
A Expression of p-p65 (Ser536) and total p65 in BMDMs from Hops+/+ and Hops−/− mice, after LPS treatment. Anti-HOPS antibody was used as control of HOPS expression. α-tubulin was used as loading control. Diagram on the right shows densitometric analysis for p-p65 levels relative to total p65 amount. B Expression of p-p65 (Ser536) and total p65 in RAW 264.7 cells transfected or not with myc-HOPS and treated with LPS for the indicated times. Anti-myc antibody was used as HOPS transfection control. α-tubulin was used as loading control. Densitometric analysis for p-p65 phosphorylation rate versus total p65 expression levels is shown on the right. C HEK 293 T cells were cotransfected with myc-HOPS and Flag-p65. HOPS was immunoprecipitated with anti-myc antibody, and co-immunoprecipitated proteins were revealed with anti-Flag and anti-myc antibodies. Total cell lysate input was revealed with anti-Flag and anti-myc antibodies. D Same as in C, except that HEK 293T cells were cotransfected with myc-HOPS and Flag-p50. E HEK 293 T cells were transfected with HOPS or an empty vector (Control) and treated or not with LPS (10 μg/ml). Endogenous p65/p50 binding was studied by protein immunoprecipitation with anti-p105/p50 antibody and WB analysis with anti-p65 antibody. Total cell lysate input was revealed with anti-p65, anti-p105/p50 and anti-HOPS antibodies. Asterisks in C, D, E indicate IgG. Arrows in E indicates p105 and p50 specific bands.
