Fig. 5.

TRIM28 S473 phosphorylation induces enhanced transcriptional activity. (A) TRIM28-knockdown HT-29 cells were reconstituted with the mock-vector or TRIM28 S473 mutants (TRIM28 S473 wild-type, TRIM28 S473A, and TRIM28 S473D). Cells were treated with TSZ for 4 h, stained, and visualized by confocal fluorescence microscopy. (green: TRIM28; red: S473 phospho-TRIM28; blue: DAPI). (B-E) Cells from (A) were treated with TSZ at the indicated times, and cell viability was determined by MTT assay (B). Cell lysates were analyzed by western blotting (C, D, and E). (F) 293 T cells were transiently transfected with Flag-TRIM28 S473 WT or the S473 mutants. After 24 h, IL-1β and CCL4 mRNA levels were measured by qPCR. (G) 293 T cells were transiently transfected with HA-p65, Flag-TRIM28 S473 WT, and various doses of Flag-TRIM28 S473D. After 24 h, luciferase activity was measured. (H) TRIM28-knockdown HeLa cells stably expressing RIPK3 were transiently transfected with Flag-TRIM28 S473 WT or S473 mutants. After 24 h, cells were treated with TNF-α for 6 h, and luciferase activity was measured