TRIM21 promotes the polyubiquitination of PP1α.

(A) IP analysis of the ubiquitination of PP1α in HEK293T cells by co-transfecting p3xFlag-CMV-PP1α with pcDNA3.1a-vector or pcDNA3.1a-TRIM21-WT or pcDNA3.1a-TRIM21-C16S as well as HA-ub for 48 h. The anti-Flag antibody was used to perform immunoprecipitation and detect Flag-PP1α, the anti-V5 antibody was used to detect V5-TRIM21-WT/C16S and the anti-HA antibody was used to detect HA-ub. GAPDH was used as an internal control. (B) Ubiquitination analysis ubiquitination of PP1α. HEK293T cells were co-transfected with p3xFlag-CMV-PP1α and pcDNA3.1a-TRIM21 as well as the indicated HA-tagged ubiquitin mutants for 48 h. The anti-Flag antibody was used to perform immunoprecipitation and detect Flag-PP1α, the anti-V5 antibody was used to detect V5-TRIM21 and the anti-HA antibody was used to detect HA-ub. GAPDH was as an internal control. (C) Ubiquitination analysis the K6-linked ubiquitination of PP1α. HEK293T cells were co-transfected p3xFlag-CMV-PP1α with pcDNA3.1a-vector or pcDNA3.1a-TRIM21-WT or pcDNA3.1a-TRIM21-C16S as well as the indicated HA-tagged ubiquitin mutants for 48 h. Immunoprecipitation was performed with anti-Flag antibody. Ubiquitination was detected by anti-HA antibody and GAPDH was used as an internal control. (D) Ubiquitination analysis the K6-linked ubiquitination of PP1α under stress. HEK293T cells pre-infected with lentivirus-sh-vector or lentivirus-sh-TRIM21 for 12 h were co-transfected p3xFlag-CMV-PP1α with HA-ub-WT or HA-ub-K6O for 36 h, then infected with VSV (MOI = 0.2) for 6 h or treated with TG (10 μM) for 12 h. Immunoprecipitation was performed with anti-Flag antibody. Ubiquitination was detected by anti-HA antibody, TRIM21 was detected by anti-TRIM21 antibody and GAPDH was used an internal control. (E) A schematic diagram of PP1α truncations. (F) Ubiquitination of C-terminus and N-terminus of PP1α in HEK293T cells by co-transfecting p3xFlag-CMV-PP1α-C-EGFP or p3xFlag-CMV-PP1α-N-EGFP with pcDNA3.1a-vector or pcDNA3.1a-TRIM21-WT or pcDNA3.1a-TRIM21-C16S as well as HA-ub for 48 h. Immunoprecipitation was performed with anti-Flag antibody. Ubiquitination was detected by anti-HA antibody, V5 was detected by anti-V5 antibody and GAPDH was used an internal control. (G-H) TRIM21 promotes the polyubiquitination of PP1α on Lys60. Mutants of only one lysine residue retained within N-terminal PP1α (G). HEK293T cells were co-transfected pcDNA3.1a-vector or pcDNA3.1a-TRIM21 with or the mutants of p3xFlag-CMV-PP1α-N for 48 h. Ubiquitination and immunoblotting were performed with the antibodies as described in (F) (H). (I) TRIM21 promotes K6-linked ubiquitination of PP1α on Lys60. HEK293T cells were co-transfected p3xFlag-CMV-PP1α-WT or p3xFlag-CMV-PP1α-K60R with pcDNA3.1a-vector or pcDNA3.1a-TRIM21-WT or pcDNA3.1a-TRIM21-C16S as well as HA-ub-K6O for 48 h. Ubiquitination and immunoblotting assays were performed with the antibodies as described in (F). Experiments were independently repeated two or three times with similar results.