Fig. 6.

CHIP promotes HMGB1 degradation via the ubiquitin proteasome pathway. A HEK293T cells were cotransfected with HA-CHIP, Flag-HMGB1, and HA-UB. The ubiquitylation level of HMGB1 was detected using an anti-HA antibody (HA-tagged UB). B EESC and 11Z cells were cotransfected with Flag-CHIP. The ubiquitylation level of HMGB1 was detected using an anti-ubiquitin antibody (UB). C EESC and 11Z cells were cotransfected with shCHIP. The ubiquitylation level of HMGB1 was detected using an anti-ubiquitin antibody (UB). D HEK293T cells were cotransfected with HA-CHIP (WT or mutants), Flag-HMGB1, and HA-UB. The ubiquitylation level of HMGB1 was detected using an anti-HA antibody (HA-tagged UB). E HEK293T cells were cotransfected with different ubiquitin mutants with HA-CHIP and Flag-HMGB1. The ubiquitylation level of HMGB1 was detected using an anti-HA antibody (HA-tagged UB). A–E After transfection for 48 h, the cells were treated with MG132 (20 μg/mL) for 8 h and then collected for ubiquitylation assays