Fig. 3

TRIM28 is required for K63-linked ubiquitination of RIPK1. (A) Ectopic HA-TRIM28 interacts with Flag-RIPK1 in 293T cells. Endogenous TRIM28 interacts with endogenous RIPK1 in CMT-167 and H1299 cells. (B) HEK293 cells were transfected with HA-TRIM28 and Flag-RIPK1 as indicated. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-ubiquitin, anti-RIPK1, or anti-HA as indicated. (C and D) RIPK1 ubiquitination is increased upon overexpression of TRIM28-WT but not the ΔR mutant. 293T cells were transfected with HA-TRIM28 WT or ΔR mutant, and the cell lysates were subjected to immunoprecipitation using anti-Flag antibodies (C) or Ni-NTA pull-down under denaturing conditions (D), followed by immunoblotting with the indicated antibodies (E) RIPK1 ubiquitination was decreased upon TRIM28 depletion. 293T cells were co-transfected with the indicated plasmids or shRNAs, and Flag-RIPK1 was immunoprecipitated and analyzed by immunoblotting. (F) H1299 cells were transfected with control or TRIM28 shRNA, and endogenous RIPK1 was immunoprecipitated and analyzed for ubiquitination (G) TRIM28 modified RIPK1 by K63-linked ubiquitination. Cell lysates prepared in (C) were immunoprecipitated with anti-Flag and blotted with anti-ubiquitin K63 and anti-ubiquitin K48