Fig. 1.

TRIM28 is a positive regulator of TRIM24 protein stability. a Equal amount of protein lysates from a panel of prostate cells were loaded for SDS-PAGE and western blot analysis was carried out to detect expression of TRIM24 and TRIM28. Tubulin was used as loading control. b TRIM28, but not other interactors, positively regulate TRIM24 protein level. Lentiviral supernatant containing pLKO control or shRNAs against top 10 TRIM24-interactors were used to infect LNCaP cells. Four days after infection, protein lysates were harvested and immunoblot performed to examine the expression of TRIM24. GAPDH was used as a loading control. c TRIM28 positively regulates TRIM24 protein. LNCaP cells expressing control (pLKO) or shTRIM28 were hormone-starved for 3 days and treated with either ethanol or 1 nM R1881 for 48 h. Protein lysates were harvested and subjected to immunoblotting analysis using indicated antibodies. d PCa cell lines expressing control (pLKO) or shTRIM28 were subjected to western blot analysis. Tubulin was used as a loading control. e TRIM24 transcript expression is not dependent on TRIM28. RNA was extracted from LNCaP cells expressing control (pLKO) or shTRIM28 and subjected to qRT-PCR analysis. Data shown is mean (± SEM, n = 3). NS: not significant. f, g TRIM28 prolongs the half-life of TRIM24 protein. LNCaP cells infected with shCtrl or shTRIM28 lentiviruses were treated with 100ug/ml Cyclohexamide and protein lysates were collected at the indicated times for western blot analysis (f). TRIM24 protein level was quantified and plotted relative to the 0 time point (g)