Fig. 3: TRIM24 interacts with and ubiquitinates VHL.

A HEK293T cells were co-transfected with HA-PHLPP1/HA-PHLPP2/HA-VHL and Flag-TRIM24/Flag-ΔRING (truncated TRIM24 mutant, lacks RING domain) plasmids and treated with or without proteasome inhibitor MG132 (10 μM) for 8 h. Whole cell lysates (Input) were derived and immunoblotted directly or after immunoprecipitation with anti-HA antibody for indicated proteins. Data are representative of three independent experiments. B Upper: In vitro ubiquitination assays. HEK293T cells transfected with GFP-TRIM24 or GFP-ΔRING for 36 h and cell lysates were immunoprecipitated using an anti-GFP antibody. The immunoprecipitants and recombinant VHL were used for in vitro ubiquitination assays. The reaction products were immunoblotted using indicated antibodies. Bottom: The levels of indicated proteins, normalized to vehicle control, were quantified (data are representative of three independent experiments). AU, arbitrary unit. Data were expressed as means ± SEM, two-tailed Student’s t-test. Data are representative of three independent experiments. C DLD1 cell lysates (input) were immunoprecipitated by anti-TRIM24 antibody, IgG control antibody, or anti-VHL antibody, respectively. Data are representative of three independent experiments. The input and the immunoprecipitants were immunoblotted for indicated proteins. D Upper: Schematic diagram of full-length human TRIM24. RING domain; B1 and B2, B-box 1 and 2; CC, coiled-coil domain; PHD, plant homeodomain; BROMO, bromodomain. Bottom: GST pull-down assays with GST, GST-fusion proteins (TRIM24 and truncated TRIM24 mutants), and His-VHL. The input and the bound proteins were immunoblotted with indicated antibodies. Data are representative of three independent experiments. E DLD1-WT/DLD1-KO-1 and SW620-WT/SW620-KO-1 cells were treated with 10 μM MG132 for 8 h. Whole cell lysates were immunoblotted for indicated proteins, with Tubulin as a loading control. Data are representative of three independent experiments. F DLD1-WT/DLD1-KO-1 and SW620-WT/SW620-KO-1 cells were treated with 100 μg/mL cycloheximide (CHX) for the indicated time periods. Whole cell lysates were derived and immunoblotted for indicated proteins. Data are representative of three independent experiments. G Representative immunoblots of indicated proteins in adjacent normal tissue and tumor tissue in patients with CRC. Shown are the representative results from three independent experiments. H, I Linear regression analysis of the levels of TRIM24 protein versus VHL (H) and β-catenin (I) in colonic epithelial cells isolated from normal and tumor tissues of patients with colorectal adenocarcinoma (n = 10). AU, arbitrary unit. Linear regression model with two-sided test was used for the statistical analysis, and multiple R-squared was used. Relationships are denoted with solid lines and fit statistics (R² and p values).