Fig. 5

BBR induces UHRF1 degradation via the ubiquitin-proteasome system pathway. a BBR affected the stability of UHRF1 in MM cells. RPMI-8266 and MM.1S cells were treated with DMSO alone, BBR (25 μM) alone for 24 h, or pretreated with DMSO or BBR for 12, 16, and 20 h, followed by addition of CHX (50 μg/mL) for additional 4, 8, and 12 h. Cell lysates were harvested and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. b, c Densitometry was utilized to quantify UHRF1 protein levels after normalization with GAPDH control to obtain percent UHRF1 degradation in RPMI-8266 and MM.1S cells. The data were presented as the mean ± SD obtained from three independent experiments. d MG132 abolished the effect of BBR on UHRF1 degradation. RPMI-8266 and MM.1S cells were treated with DMSO alone, BBR (25 μM) alone for 24 h, or pretreated with DMSO or BBR for 20 h, followed by addition of MG132 for additional 4 h. Cell lysates were harvested and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. e RPMI-8266 cells were subsequently treated with BBR (25 μM for 24 h) prior to harvesting. The proteins modified by ubiquitination were purified from cell extracts using anti-UB beads and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. f RPMI-8266 cells were transiently transfected with HA-Ub constructs, and endogenous UHRF1 proteins were immunoprecipitated from BBR-treated or BBR-untreated RPMI-8266 cells (25 μM for 24 h). Immunoprecipitates were harvested and subjected to western blotting with anti-UHRF1, anti-FK2, and anti-GAPDH antibodies