Fig. 4: USP51 binds and stabilizes DGCR8.

a LM2 cells were treated with 10 μM MG132, irradiated with 8 Gy X-ray, and collected 6 h later. Lysates were immunoblotted with antibodies against DGCR8 and β-actin. b Parental and radioresistant LM2 cells were treated with 100 μg/ml cycloheximide (CHX). Cells were collected at different time points and immunoblotted with antibodies against DGCR8 and β-actin. c HEK293T cells were co-transfected with MYC-DGCR8 and HA-ubiquitin (Ub) or its lysine-specific mutants (K48, K63, K48R, or K63R), followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. Cells were treated with IR (8 Gy), followed by treatment with 10 μM MG132 for 6 h. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. d SFB-tagged DUBs were individually co-transfected with MYC-DGCR8 into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. e HEK293T cells were co-transfected with MYC-DGCR8, HA-ubiquitin, and a candidate DUB. After treatment with MG132 for 6 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. f Immunoblotting of DGCR8, USP36, USP51, and β-actin in LM2 cells with overexpression or knockdown of USP36 or USP51. g HEK293T cells were co-transfected with MYC-DGCR8 and SFB-tagged USP36 or USP51. After 48 h, cells were lysed, immunoprecipitated with anti-MYC beads, and immunoblotted with antibodies against FLAG and MYC. h Co-IP of endogenous DGCR8 with endogenous USP36 and USP51. DGCR8 was immunoprecipitated from LM2 cells and immunoblotted with antibodies against USP36, USP51, and DGCR8. i USP51 binds DGCR8 in vitro. Left panel: SFB-GFP or SFB-USP51 was retained on S-protein beads and incubated with purified MBP-DGCR8. The bound proteins were eluted by boiling in Laemmli buffer and immunoblotted with antibodies against MBP and FLAG. Right panel: purified SFB-GFP, SFB-USP51, and MBP-DGCR8 proteins were analyzed by SDS-PAGE and Coomassie blue (CB) staining. j Upper panel: LM2-R cells stably infected with USP51 shRNA were treated with 100 μg/ml CHX for the indicated times. Lysates were subjected to immunoblotting with antibodies against DGCR8, USP51, and β-actin. Lower panel: DGCR8 levels were quantitated and normalized to β-actin. Source data are provided as a Source Data file.