Fig. 1: USP5 removes K11-linked polyubiquitination from YTHDF1 and inhibits its degradation.
From: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation
a Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) from HEK293T cells transfected with GFP-Ub and Flag-YTHDF1. b YTHDF1 polyubiquitination could largely be detected in cells transfected with indicated constructs. c IB analysis of WCL and His immunoprecipitate from HEK293T cells transfected with indicated constructs. d PLC/PRF/5 cells were immunoprecipitated with either anti-USP5 or anti-YTHDF1 antibody and then analyzed by IB. e IB and IP products analysis of USP5-YTHDF1 interaction in HEK293T cells expressing HA-USP5 WT or the indicated truncated YTHDF1 mutants. f, g IB analysis of YTHDF1 levels in HEK293T cells expressing HA-USP5 (DNA content of 250 ng or 500 ng) or indicated plasmids. h IB and QRT-PCR analysis of YTHDF1 from Hepa1-6 cells with Usp5 knockout. n = 3. i, j IB analysis of WCL from Hepa1-6 cells with the depletion of Usp5 or HEK293T cells transfected with indicated constructs for 36 h. Cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 levels was quantified by the ImageJ software. k IB analysis of WCL and IP products from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. l Effects of Usp5 knockout in Hepa1-6 cells on Ythdf1 K11-linked polyubiquitination were evaluated by IB. m Effects of WP1130 on USP5-mediated YTHDF1 K11-linked polyubiquitination. Cells expressing indicated plasmids were treated with different doses of WP1130. n IB analysis of proteins labeled with puromycin using anti-puromycin antibody upon Usp5 depletion with or without expressing Ythdf1. o, p Assessment of subcutaneous tumor formation from PLC/PRF/5 cells after depletion of USP5, or YTHDF1, or stably expressing YTHDF1 with endogenous USP5 knockdown. Tumor weight was measured at the endpoint of the study. Tumor growth was measured at the indicated time points. n = 5. *p < 0.05, t-test. q Kaplan-Meier analysis revealed a relationship between YTHDF1 and USP5 expression and overall survival in HCC patients. All data are presented as mean ± SEM. All IB data are representative of three independent experiments. Source data are provided as a Source Data file.
