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. 2024 Jan 5;223(3):e202306013. doi: 10.1083/jcb.202306013

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© 2024 Endo et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S5. — Colocalization of p62 and Keap1 on endosomes in USP8-depleted cells. (A) Total cell lysates from parental HeLa cells and HeLa cells stably expressing FLAG-p62 WT, dUBA, and VVV were immunoblotted with the indicated antibodies. (B) HeLa cells stably expressing FLAG-p62 WT, dUBA, and VVV were transfected with the indicated siRNAs. Cells were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (C) HeLa cells were transfected with the indicated siRNAs. Endogenous p62 immunoprecipitated with anti-p62 antibody and total cell lysates were immunoblotted with the indicated antibodies. (D) HeLa cells stably expressing FLAG-p62 WT were transfected with the indicated siRNAs. FLAG-p62 immunoprecipitated with anti-FLAG antibody under denaturing conditions and total cell lysates were immunoblotted with the indicated antibodies. (E–G) HeLa cells stably expressing FLAG-Keap1 were transfected with the indicated siRNAs. Cells were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (H and I) Total RNA from HeLa cells transfected with the indicated siRNAs was analyzed by qRT-PCR. SOD2 (H) and HO1 (I) expression levels were normalized to actin mRNA levels, and expression levels in cells treated with control siRNA were set to 1. Individual values, mean, and SE of the mean of relative mRNA levels are shown. Means ± SE were calculated from four biological replicates. ***P < 0.001, ****P < 0.0001 (one-way ANOVA with Dunnett’s test). (J) Parental HeLa cells and HeLa cells stably expressing siRNA-resistant Myc-USP8 WT and C786A were transfected with the indicated siRNAs. Total RNA from cells was analyzed by qRT-PCR. SOD2 expression levels were normalized to actin mRNA levels, and expression levels in cells treated with control siRNA were set to 1. Individual values, mean, and SE of the mean of relative mRNA levels are shown. Means ± SE were calculated from four biological replicates. ***P < 0.001 (one-way ANOVA with Dunnett’s test). Source data are available for this figure: SourceData FS5.