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. 2021 Aug 17;220(10):e202103176. doi: 10.1083/jcb.202103176

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© 2021 Ferreira and Carvalho

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 S2. — Protein levels of Pex30 binding partners are dependent on Pex30 availability. (A) Steady-state levels of endogenously tagged Pex28, Pex29, Pex30, and Pex32 in cells with the indicated genotype. Whole-cell extracts were analyzed as in Fig. 1 D. Pex28-13MYC, Pex29-V5, Pex32-3HA, Pex30, and Dpm1 (used as loading control) were detected with anti-MYC, anti-V5, anti-HA, anti-Pex30, and anti-Dpm1 antibodies, respectively. IB, immunoblot. (B) Quantification of protein levels in cells in the indicated genotype. The levels of Pex30 family proteins determined as in A were compared with WT levels. The average of three independent experiments is shown. Bars represent SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, P > 0.5). (C) Pex32 interaction with Pex30 is dependent on Pex28. Crude membrane fractions of cells with the indicated genotypes and expressing endogenous Pex28, Pex29, and Pex32 fused respectively, to MYC, V5, and HA epitope tags or untagged proteins as control were detergent solubilized, and extracts were subjected to immunoprecipitation (IP) with anti-MYC, V5, or HA antibodies. Eluted proteins were analyzed by Western blotting.