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. 2024 Aug 20;43(39):2914–2926. doi: 10.1038/s41388-024-03121-1

Fig. 4. 6b promotes BRD4 ubiquitination and degradation dependent on CRBN.

Fig. 4

A HCC1937 and HCC1806 cells were treated with indicated 6b and at the same time MG132 (3 μM) were added for 8 h. Cell lysates were collected for WB. B HEK293 cells were co-transfected with plasmids expressing 3×Flag-BRD4 and HA-Ub for 2 days. Flag-BRD4 was immunoprecipitated with anti-Flag M2 beads under denaturing conditions. Ubiquitinated BRD4 proteins were detected by anti-BRD4 antibody. C HCC1937 and HCC1806 cells were treated with 6b (0-0.01-0.1 μM) for 48 h and then MG132 (20 μM) were added for the next 8 h. Cell lysates were collected for WB. D CRBN was knocked down by two different siRNAs in HCC1937 and HCC1806 cells, as analyzed by RT-qPCR. E. CRBN was knocked down by two different siRNAs in HCC1937 and HCC1806 cells for 48 h. 48 h later, cells were treated with 6b for indicated concentration, and cell lysates were collected for WB. F. CRBN knockdown by two different siRNAs abrogated the inhibitory effects of 6b in HCC1937 and HCC1806 cells. Cell viability was measured by the SRB assay. *p < 0.05, **p < 0.01. ***p < 0.001. t-test.