Fig. 6: PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL.
A HOIP−/− or HOIP reconstituted MEFs were treated with TNFα for indicated periods time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-TNFR1 antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. B PP6+/+ and PP6−/− L929 cells with Flag-RIPK1 stable expression were treated with TNFα for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-Flag beads. The isolated complex I was eluted with urea lysis buffer. The eluted complex I was further immunoprecipitated with chain specific M1 or K63 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. C Flag-RIPK1 was co-expressed with LUBAC (HA-HOIP/Myc-HOIL/Flag-Sharpin) or HA-PP6 in 293T cells for 20 h. Cells were lysed with urea lysis buffer and immunoprecipitated with chain specific M1 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. D Flag-tagged c-FLIPL was co-expressed with LUBAC (HA-HOIP/Myc-HOIL/Flag-Sharpin) or HA-PP6 in 293T cells for 20 h. Cells were treated with PS341 8 h after transfection. Cells were lysed with urea lysis buffer and immunoprecipitated with chain specific M1 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. Concentrations of reagents used: TNFα (T), 50 ng/ml; PS341, 100 nM.
