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. 2022 Aug 29;119(36):e2119854119. doi: 10.1073/pnas.2119854119

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Copyright © 2022 the Author(s). Published by PNAS.

This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 1. — USP13 interacts with ZHX2 and promotes USP13 protein stability. (A) Representative immunoblots of immunoprecipitations (IPs) and whole-cell extracts (WCEs) from 293T cells transfected with FLAG and HA double-tagged DUB cDNA library (4 µg plasmid per p60 plate) as indicated and then treated with 10 μM MG132; FLAG-HA-GFP is the negative control, and FLAG-VHL is the positive control. (B) Immunoblots of the lysates from UMRC-2 transfected with siRNAs of some candidate DUB proteins. (C and D) Immunoblots of the WCE and IP from 293T transfected with FLAG-HA-USP13, FLAG-HA-GFP, FLAG-VHL, HA-ZHX2, and HA-GFP as indicated. (E and F) Immunoblots of the WCE and IP from 293T and 786-O cells as indicated. (G and H) Immunoblots of the lysates from 293T and HKC transfected with FLAG-HA USP13, FLAG-HA-GFP, and FLAG/HA-VHL as indicated. (I and J) Immunoblots of lysates from 293T and HKC cells transfected with EV, WT, or CD form of USP13 as indicated. (K) Immunoblots of lysates from HKC cells transfected with EV, WT, or CD form of USP13 and then treated with 10 μg/mL CHX for the indicated time. (L) Quantification of ZHX2 expression level shown. (M) Immunoblots of WCE or IP from UMRC-2 or 786-O cells infected with FLAG-USP13 or empty vectors and then treated with 10 μM MG132 as indicated. (N) Immunoblots of WCE or IP from 293T cells transfected with indicated plasmids and then treated with 10 μM MG132 as indicated. Error bars, SEM; **P < 0.01; ***P < 0.001; MW, molecular weight.