Fig. 4

TRIM15 promotes Keap1 ubiquitination and degradation. A Immunoblot analysis of Keap1, Flag-TRIM15, and β-actin in HEK293 cells transfected with expression vector for Flag-TRIM15 or with empty vector, with or without treatment of 10 μM MG132. Mutations of Flag-TRIM15-ΔRING impaired the ability of TRIM15 to degrade Keap1 protein in HEK293 cells. B HEK293 cells were transfected with the indicated plasmids. The cells were treated with 50 μg/ml CHX for indicated time periods. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. C H1299 or H1650 cells were transfected with the indicated plasmids, treated with 50 μg/ml CHX, harvested at different time points, and then immunoblotted with using the indicated antibodies. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. D HEK293 cells were transfected with Flag-TRIM15, Flag-TRIM15-ΔRING, HA-Keap1, and His-ubiquitin plasmids, and the cell lysates were subjected to immunoprecipitation using anti-HA antibodies (left panel) or Ni–NTA pull-down (right panel) under denaturing conditions, followed by immunoblotting with the indicated antibodies. Overexpression of wild-type but not the mutant TRIM15-ΔRING promoted ubiquitination of Keap1. E Knockdown of endogenous TRIM15 decreased the ubiquitination of HA-Keap1 in HEK293 cells analyzed by in vitro ubiquitination assays. F Cell lysates prepared in D were immunoprecipitated with anti-HA. knockdown of TRIM15 inhibits K48-linked but not K63-linked ubiquitination of HA-Keap1