Fig. 2: USP5 deubiquitinates YTHDF1 on multiple lysine residues to confer its oncogenicity.

a IB analysis of WCL and anti-Flag IPs from HEK293T cells transfected with indicated constructs and treated with the 20 μM MG132 for 8 h. b In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 WT and indicated truncated YTHDF1 mutants in the presence or absence of ectopic USP5 expression. Cells were treated with 20 μM MG132 for 8 h. c IB analysis of the protein levels of the indicated truncated YTHDF1 mutants in HEK293T cells expressing increasing amounts of HA-USP5. d IB analysis of WCL and IP derived from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. e IB analysis of WCL derived from HEK293T cells transfected with indicated constructs. 36 h post-transfection, cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 protein abundance was quantified by the ImageJ software. f In vivo ubiquitination assays of WCL and anti-Flag IPs derived from HEK293T cells transfected with plasmids expressing the indicated proteins. Cells were treated with 20 μM MG132 for 8 h. g In vivo ubiquitination assay of USP5 knockout in PLC/PRF/5 cells transfected with WT Flag-YTHDF1 or indicated mutant constructs. YTHDF1 polyubiquitination was evaluated by IB analysis. Cells were treated with 20 μM MG132 for 8 h. h Colony formation assays of PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. n = 3 per group. **p < 0.01. t-test. i, j Assessment of subcutaneous tumor formation from PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. Tumor weight was measured at the endpoint of the study. In vivo tumor growth was measured at the indicated time points and tumors were dissected at the endpoint. n = 6 per group. **p < 0.01, t-test. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a Source Data file.