Fig. 7

TRIM18 induces ubiquitination of PPM1A by K63 linkage. a Immunoblot (IB) analysis of TRIM18 (fourth blot), PPM1A (fifth blot) and β-actin (bottom blot) in WT and TRIM18 KO BMDM, the abundance (top blot), total ubiquitination (second blot), and K63-mediated ubiquitination (third blot) of PPM1A in those cells, infected for 4 h with CVB3 or adenovirus at an MOI of 5, assessed after immunoprecipitation with anti-PPM1A antibody. b Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot), and K63-linked ubiquitination (third blot) of Myc-tagged PPM1A in HEK293T cells transfected with empty vector or expression vector for full length HA-tagged TRIM18 (HA-T18 full), truncation T18-∆SPRY (losing binding site of PPM1A), assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-HA (fourth blot), anti-Myc (fifth blot) and anti-β-actin (bottom). c Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot), and K63-linked ubiquitination (third blot) of Myc-tagged PPM1A in HEK293T cells transfected with HA-TRIM18 and Myc-PPM1A wild-type (WT) and its mutations including K9RK12R, K118RK119R, K156R, K296R, K303RK304R, and K354R, assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-Myc (fourth blot), anti-HA (fifth blot) and anti-β-actin (bottom). d Luciferase assay in HEK293T cells transfected with IFN-β reporter, Flag-tagged plasmid expressing TBK1 along with Myc-tagged empty vector or PPM1A wild-type (WT) and its mutations including K9RK12R, K118RK119R, K156R, K296R, K303RK304R, and K354R. Renilla luciferase RL-TK was used as the internal control. e Immunoblot analysis (with anti-Myc) of the abundance (top), and K63-linked ubiquitination (second blot) of Myc-tagged PPM1A in HEK293T cells transfected with HA-TRIM18, Flag-TBK1 and Myc-PPM1A wild-type (WT) and its ubiquitination losing mutant K9/12/296/354R, assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-phosphorylated (p-) TBK1 (third blot), anti-TBK1 (fourth blot) and anti-β-actin (bottom). f Luciferase assay in HEK293T cells transfected with IFN-β reporter, Flag-tagged plasmid expressing TBK1 along with Myc-tagged empty vector or PPM1A wild-type (WT) and its ubiquitination losing mutant K9/12/296/354R. Renilla luciferase RL-TK was used as the internal control. NS, not significant (p > 0.05), ***p < 0.001, and p value was calculated by unpaired two-tailed Student’s t test. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments