Skip to main content
. 2020 Oct 23;11:5362. doi: 10.1038/s41467-020-19202-0

Fig. 5. ATM kinase promotes the activity of USP52 to regulate the DDR.

Fig. 5

a HEK293T cells transfected with FLAG–USP52 were treated with DMSO or 25 μM Ku55933 for 2 h prior to IR treatment. Harvested cells were immunoprecipitated with anti-FLAG agarose beads. After untreated or treated with lambda protein phosphatase, blots were probed with pSQ/TQ antibody. b HEK293T cells transfected with indicated USP52 constructs were harvested after IR and then immunoprecipitated with anti-FLAG agarose, blots were probed with indicated antibodies. c Ubiquitinated CtIP was incubated with purified FLAG-WT USP52 or the S1003A mutant before or after IR to perform deubiquitination reaction assay in vitro, and then blotted with the indicated antibodies. d, g Control or USP52-depleted U2OS cells were transfected with indicated USP52 truncates before treated with 2 Gy IR for 2 h or 5 Gy IR for 5 h. RPA2 and Rad51 focus formation were detected by immunofluorescence (d, f) and quantified (e, g). Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 μm. h Control or USP52-depleted HEK293T cells transfected with indicated USP52 constructs together with HR reporter were harvested for HR assay. Error bars represent SEM from three independent experiments. i USP52-depleted HEK293T cells were transfected with indicated USP52 constructs for 24 h before treated with or without IR. Harvested cells were then immunoprecipitated with nickel (His) beads and blotted with indicated antibodies. j Purified WT USP52 or the S1003A mutant was incubated with ATM for in vitro kinase assay, and then ATM-phosphorylated USP52 constructs were used for in vitro deubiquitination reaction with ubiquitinated GFP-CtIP. k Control or USP52-depleted HEK293T cells transfected with indicated USP52 constructs were harvested and immunoprecipitated with anti-FLAG agarose, and then subjected to blot with the indicated antibodies. l, m Control or USP52-depleted U2OS cells stably expressing indicated USP52 constructs were treated with IR or PARPi for 2 weeks. Cell viability was assessed using colony formation assay. Error bars represent SEM from three independent experiments.