Fig. 2: USP2 interacts with CD47 and deubiquitinates CD47.

a IB analysis of HEK293T WCL and anti-HA immunoprecipitates (IPs). HEK293T cells were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. b IB analysis of glutathione S-transferase (GST) pull-down protein mixture from HEK293T cell lysates that overexpressed CD47-cHA incubated with bacterially purified recombinant GST or GST-USP2 protein. c IB analysis of GST pull-down products from HEK293T cell lysates that overexpressed Flag-USP2 incubated with bacterially purified recombinant GST or GST-CD47 protein. d Schematic representation of WT and truncations of USP2, including the N-terminal region of amino acid (aa) 1–266 and C-terminal domain of aa267–605. e IB analysis of WCL and anti-HA IPs obtained from HEK293T cells, which were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. f IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA incubated with bacterially purified recombinant GST, GST-USP2 WT, and GST-USP2 truncations. g IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of Flag-USP2 WT and truncations incubated with bacterially purified recombinant GST-CD47 protein. h Schematic diagram of CD47 WT and its various deletion mutants. i IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA WT and deletion mutants incubated with bacterially purified recombinant GST-USP2 protein. j, k IB analysis of WCL and IPs derived from H1975 (j) and PC9 (k) cells. l, m IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. n IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 2 µM ML364 for 16 h and 10 µM MG132 for 12 h before harvesting. o, p IB analysis of WCL and IPs derived from lysates of H1975 (o) and PC9 (p) cells using indicated K48-Ubi antibodies. Cells were treated with 20 µM MG132 for 6 h before harvesting. q, r IB analysis of WCL and anti-CD47 IPs derived from H1975 (q) or PC9 (r) cells stably expressing shUSP2 or shGFP, respectively. Cells were treated with 20 µM MG132 for 6 h before harvesting. n = 3 biologically independent experiments for (a, b, c, e, f, g, and i–r). Source data are provided as the Source Data file.