Fig. 5.

CHIP downregulates HMGB1 protein stability. A HEK293T overexpressed Flag-CHIP (0, 0.5 or 1 μg) and HA-HMGB1 protein. Immunoblotting experiments were performed. B HEK293T overexpressed Flag-CHIP (WT or mutants) and HA-HMGB1 protein. Immunoblotting experiments were performed. C Overexpression or knockdown CHIP with Flag-CHIP or shRNA in 11Z cells, respectively. Immunoblotting experiments were performed. D Overexpression or knockdown CHIP with Flag-CHIP or shRNA in EESC cells, respectively. Immunoblotting experiments were performed. E 11Z cells were transfected with Flag-CHIP for 48 h and then treated with MG132 (20 μg/mL) for 8 h. Immunoblotting experiments were performed. F HEK293T cells with overexpression of Flag-CHIP and HA-HMGB1 were treated with CHX (100 μg/mL) for indicated time. Immunoblotting experiments were performed. (Representative western blot was quantified by ImageJ software, and statistics were normalized to β-actin. All data represent mean ± SEM. Statistical significance was analyzed with Student’s t test and one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)