Fig. 4
From: Deubiquitinase USP13 dictates MCL1 stability and sensitivity to BH3 mimetic inhibitors
USP13 interacts with and deubiquitinates MCL1. a 3 × FLAG-tagged USP13 was transfected into HEK293T cells. Cells were treated with MG-132 for 8 h and USP13 was immunoprecipitated using an anti-FLAG antibody. Co-immunoprecipitated MCL1 was detected using an anti-MCL1 antibody. b Endogenous USP13 and MCL1 were immunoprecipitated from A549, HEY and SW-1573 cells using an anti-USP13 antibody or an anti-MCL1 antibody respectively. Co-immunoprecipitated endogenous MCL1 or USP13 was analyzed by western blotting. c Left: Purified His-MCL1 and His-USP13 proteins were co-incubated and immunoprecipitated with an anti-USP13 antibody. Co-immunoprecipitated MCL1 was detected using an anti-MCL1 antibody. Right: FLAG-USP13 or FLAG-USP13-C345A was transfected into HEK293T cells and immunoprecipitated using an anti-FLAG antibody. Co-immunoprecipitated MCL1 was detected using an anti-MCL1 antibody. d A schematic of full-length USP13 (FL), full-length MCL1 (FL) and their deletion mutants (M1, M2, U1, U2). e USP13 was co-transfected with the MCL1 deletion mutants, and MCL1 was co-transfected with the USP13 deletion mutants. Interactions were analyzed using the co-immunoprecipitation assay. f MCL1 was co-transfected with FLAG-USP13 or the FLAG-USP13-C345A mutant, with or without HA-ubiquitin. The ubiquitinated MCL1 was measured using an in vivo ubiquitination assay. g Purified His-MCL1 and His-USP13 proteins were co-incubated in the ubiquitination system. The ubiquitinated MCL1 was measured using an in vitro ubiquitination assay
