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. 2021 Jan 4;12:51. doi: 10.1038/s41467-020-20259-0

Fig. 5. USP47 interacts with YB-1 and protects it from proteasomal degradation.

Fig. 5

a Immunoprecipitation with USP47 antibody in K562 cells, the proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue (G250). b The interaction between endogenous USP47 and YB-1 was analyzed by western blot in K562 and primary CML cells. c Full-length or several deletion constructs of GFP-YB-1 was co-transfected with Flag-USP47 in HEK293T cells. Their interactions were examined by co-IP with anti-GFP antibody and by western blot with anti-Flag antibody. d GFP-YB-1-FL/GFP-YB-1-S2/GFP-YB-1-S3 and HA-ubiquitin plasmids were co-transfected with or without Flag-USP47 into HEK293T cells. YB-1 was co-IP with anti-GFP antibody, and its ubiquitination was measured with Ub antibody by western blot. e Normal and CML BM mononuclear cells were lysed and co-IP with YB-1 antibody, YB-1 ubiquitination was detected with Ub antibody. f Flag-USP47, GFP-YB-1, and GFP-YB-1-mu (K137A, K164A, and K170A) were transfected in HEK293T cells. Their interactions were examined by co-IP with anti-Flag antibody and measured by western blot with anti-GFP antibody. g GFP-YB-1 or GFP-YB-1-mu was transfected with HA-Ub in HEK293T cells. YB-1 ubiquitination in transiently transfected cells was analyzed by co-IP with anti-GFP antibody and western blot with Ub antibody. h USP47 plasmids and empty vectors were transfected into K562 cells. The transfected cells were treated with cycloheximide (CHX, 10 μM) at different times. The indicated proteins were determined by western blot. i, j USP47 knockdown cells or Usp47−/− MEFs were treated with cycloheximide (CHX, 10 μM) at different times together with the control cells, and the indicated proteins were determined by western blot. k Control and USP47 stably knockdown K562 cells were treated with vehicle and MG132 (10 μM) for 4 h. The proteins were then extracted and subjected to western blot. l YB-1 expression in Usp47−/− MEFs was measured by western blot after reintroduction of USP47. Source data are provided as a Source Data file.