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. 2023 Sep 18;120(39):e2305092120. doi: 10.1073/pnas.2305092120

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Copyright © 2023 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

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Fig. 3. — BRWD3 promotes KDM5 ubiquitination and degradation. (A) Denaturing IPs from Drosophila S2 cells cotransfected with HA-tagged KDM5 and/or FLAG-tagged ubiquitin in BRWD3-V5 cotransfected (OE), wild-type (WT), or BRWD3 RNAi (KD) conditions. (B) Immunoprecipitates from denaturing IPs were blotted with antibodies specific for K48- or K63-linked polyubiquitin chains together with anti-HA antibody. (C) S2 cells were transfected with plasmids expressing KDM5-HA and treated with cycloheximide (CHX) only or together with the proteasome inhibitor MG-132. After the indicated time with CHX treatment, western blots with anti-HA antibody were performed to measure the KDM5-HA level. (D) Similar as C, but with either BRWD3-RNAi or GFPi-RNAi treatment. (E) Quantification of the KDM5-HA level from three biological replicates of (D) at the indicated time points normalized to the total loaded protein level. (F) Similar to C in S2 cells with either GFPi-RNAi, Cul4-RNAi, or treatment with the Cullin inhibitor (MLN4924). (G) Quantification of the remaining KDM5-HA levels from three biological replicates of F at the indicated time points.