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. 2023 Oct 30;120(45):e2306395120. doi: 10.1073/pnas.2306395120

Fig. 1.

Fig. 1.

USP15 controls the stability of NF proteins. (A) Global proteome analysis of WT and USP15-KO 293FT cells. The threshold of fold changes was set to more than a twofold increase or decrease, and the –Log10 P-value was above 2; two biological and technical replicate samples were subject to mass spectrometric analysis. (B) Validation of proteomic data. WT and USP15-KO 293FT cells were treated with different concentrations of MLN4924 (0, 1, and 2 µM) for 8 h. Cell extracts were analyzed by immunoblotting (IB) with the indicated antibodies. (C) Quantification of NEFL and INA proteins from (B). (D) CHX experiment. USP15+/+ and USP15−/− 293FT cells were treated with cycloheximide (CHX, 50 µg/mL) at the indicated times. Cell extracts were analyzed by IB. (E) Quantification of NEFL and INA proteins from (D). (F) USP15-KO 293FT cells were transiently transfected with empty vector (EV, control), WT USP15Flag, or C298A-USP15Flag mutant plasmid. After 40 h, cells were treated with CHX for the indicated times. Cell extracts were analyzed by IB with the indicated antibodies. (G) Quantification of NEFL, INA, and GS proteins from (F). The relative ratios of indicated proteins:Actin, normalized to time 0, are shown. (H) USP15+/+ and USP15−/− 293FT cells were treated with bortezomib (Bort, 1 µM) for 7 h. Total ubiquitylated proteins were purified using TUBE2-agarose. Bound fractions and input were analyzed by IB. (I) USP15-KO 293FT cells were transiently transfected with NEFLMYC in the presence of EV, WT USP15Flag, or C298A-USP15Flag mutant plasmid. After 40 h, cells were treated with 2 µM bortezomib for 6 h, followed by cell lysis and MYC IP under denaturing conditions. The bound fractions and input were analyzed by IB with antibodies against ubiquitin (Top), MYC, Flag, and Actin. (J) In vitro deubiquitylation assay. USP15−/− 293FT cells were treated with 1 µM Bort for 7 h. Total ubiquitylated proteins, purified using TUBE2, were treated with recombinant (r)USP15 and then analyzed by IB with anti-NEFL and anti-USP15 antibodies. (Ub)n, polyubiquitin. The results shown are representative of three independent experiments (B, D, F, and H–J).