Fig. 3: TRIM25 interacts and ubiquitinates Keap1.

a Identification potential substrates of TRIM25 related to ER stress by liquid chromatography-tandem mass spectrometry. b The Keap1 peptides identified through mass spectrometry are shown. c, d Co-immunoprecipitation (co-IP) assay analyzes the interaction of endogenous TRIM25 and Keap1 in HCT116 (c) and Huh7 (d) cells, treated with or without TG (1 μM) for 12 h. e In vitro GST pulldown assay analysis of the interaction of TRIM25 (F-TRIM25). Asterisks indicate the coomassie blue staining of GST and GST-Keap1. f, g HCT116 cells infected with shRNA lentivirus as indicated with treatment of TG (1 μM) for 6 h and then treated with CHX. Representative western blot (f) and the corresponding quantified graph (g) are shown. h, i Western blot analysis the level of Keap1 in HCT116 cells treated with or without TG (1 μM) for 12 h (h), and the above cells treated with TG (1 μM) simultaneously with MG132 (4 μM) or CQ (50 μM) for 12 h (i). j The RING zinc-finger (R), B-Box, coiled-coil (CC) and PRY/SPRY (PS) domain of TRIM25 are indicated. k Huh7 cells overexpressing full-length and TRIM25 truncates were immunoprecipitated with the indicated antibody. l TRIM25 and its mutant (TRIM25-2EA), the conserved Glu9 and Glu10 were mutated to Ala. m, n HCT116 cells overexpressing the indicated constructs with treatment of TG (1 μM) for 6 h. Representative western blot (m) and the corresponding quantified graph (n) are shown. o HCT116 cells transfected with the plasmids as indicated and treated with TG (1 μM) for 12 h. Immunoblot analysis of the cell lysates and Keap1-IP with the indicated antibodies. p The broad-complex, tramtrack and bric a brac (BTB) domain, intervening region (IVR) and the double glycine repeat (DGR) or Kelch repeat of KEAP1 were indicated. q, r Huh7 cells were transfected with the indicated plasmids. Immunoblot analysis of the HA-IP and cell lysates (q), and the ubiquitination of wide-type and Keap1mutaions with the indicated antibodies (r). For g and n, data represent the mean ± SEM (n = 3). Statistical significance was assessed using two-tailed Student’s t-tests. n.s. not significant.