Extended Data Fig. 1: USP15 interacts with PARP1 in a PAR-independent manner.
(a) Screening of putative PARP1-interacting DUBs using 60 vectors encoding different DUBs. Vectors encoding HA-tagged DUBs and GFP-tagged PARP1 were cotransfected into HEK293 cells. After 24 h, cells were harvested for a co-IP assay with beads coated with GFP nanobodies prior to Western blot analysis with the indicated antibodies. (b, c) Analysis of PARP1 protein levels in USP1- or USP21-depleted cells treated with cycloheximide (CHX). HEK293 cells with depletion of USP1 (b) or USP21 (c) were treated with CHX at a concentration of 100 μg/mL and harvested for Western blot analysis at the indicated time points. (d) PARP1 mRNA levels did not change in USP15-depleted cells treated with CHX. HEK293 cells with USP15 depletion were treated with CHX at a concentration of 100 μg/mL and harvested for total RNA extraction and quantitative PCR experiments. n = 3 technical replicates. (e-f) USP15 interacted with PARP1 in a PAR-independent manner. HEK293 cells were treated with the PARP1 inhibitors 1 μM olaparib (e) or veliparib (f) for 24 h before being harvested for IP and Western blot analysis. (g) The catalytically dead PARP1 mutant retained its interaction with USP15. Plasmids encoding Flag-tagged PARP1-WT or PARP1-E988K were transfected into HEK293 cells and co-IP experiments was performed with magnetic beads coated with anti-Flag antibodies, and Western blot analysis was performed with the indicated antibodies. (h) Analysis of the interaction between USP15 and PARP1 on chromatin or in nucleoplasm. All experiments were repeated three times independently with similar results; data of one representative experiment are shown.
