Extended Data Fig. 2: USP15 stabilizes PARP1 to regulate BER.
(a) Cells with USP15 depletion were transfected with a vector encoding ubiquitin and the control vector, USP15-WT or the catalytically dead USP15-C269A mutant. Cells were supplemented with 10 μM MG132 for 12 h, and co-IP experiments with antibodies against PARP1 were performed 24 h post transfection prior to Western blot analysis with the indicated antibodies. (b) USP15 deubiquitinated PARP1 in vitro. (c) The identification of E3 ubiquitin ligases that modify PARP1 after USP15 loss. The indicated E3 ubiquitin ligases were knocked down in USP15-depleted HEK293 cells, and co-IP experiments were then performed with an antibody against PARP1, followed by Western blot analysis against K48-linked ubiquitin. (d) Analysis of RNF144A, IDUNA, RNF168, CHFR and TRIP12 levels in the indicated factor depleted HEK293 cells. (e) Co-IP analysis of the PARP1-USP15 interaction in MMS-treated HEK293 cells. HEK293 cells were treated with MMS at a concentration of 500 μM and incubated for 2 h before being harvested for co-IP with an antibody against PARP1. The precipitated proteins were then further treated with λ phosphatase prior to Western blot analysis with the anti-USP15 antibody. (f) Co-IP analysis of the PARP1-USP15 interaction in HEK293 cells treated for 6 h with MMS at different concentrations. Cells were harvested for co-IP with an antibody against PARP1. The precipitated proteins were then further treated with λ phosphatase prior to Western blot analysis with the anti-USP15 antibody. (g) Western blot analysis of the PARP1 protein level in control with USP15-depleted HEK293 cells. Cells were treated with MMS at the indicated concentrations for 6 hours before being harvested for Western blot analysis. Data are expressed as mean ± s.d., n = 3 independent biological samples. (h) Analysis of H2AX PARylation in MMS treated control and USP15 depleted cells. HEK293 cells were treated with MMS at a concentration of 400 μM and incubated for 6 h before being harvested for co-IP experiments with an antibody against PAR. All experiments were repeated three times independently with similar results; data of one representative experiment are shown.
