Fig. 2: TRIM11 interacts with and poly-ubiquitinates KDM5C.
From: Regulation of KDM5C stability and enhancer reprogramming in breast cancer
A 293T cells were transfected with indicated plasmids for 36 h and treated with 10 μM MG132 for 12 h. Co-IP was performed with anti-Flag and anti-HA antibody. B GST-TRIM11 was expressed and purified from bacteria. HA-KDM5C was expressed in 293T cells and lysates were subject to GST pulldown assay. C,D MDA-MB-231 cells treated with MG132 were used for co-IP with KDM5C and TRIM11 antibodies, followed by western blot analysis. E–G 293T cells were transfected with HA-KDM5C (E, F)/HA-KDM5C-G (G), F-TRIM11 and Myc-Ub (WT/K48/K63) plasmids for 36 h followed by MG132 (10 μM) treatment for 12 h. Cell lysates were subjected to immunoprecipitation with anti-HA followed by western blot with anti-Myc. H In vitro ubiquitination assay was performed in the presence of Ub, E1, E2(UbCH5b), bacteria-purified GST-TRIM11 and 293T-purified Flag-KDM5C. I 293T cells were transfected with Myc-KDM5C, HA-Ub, F-TRIM11(WT) or F-TRIM11(C16AC19A). Ubiquitination of KDM5C was measured. J Flag-KDM5C, F-TRIM11(WT) and F-TRIM11(C16AC19A) were expressed in 293T cells and western blotting was performed as indicated.
