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. 2021 May 26;220(8):e202009092. doi: 10.1083/jcb.202009092

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© 2021 Abudu et al.

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Figure 1. — p62 interacts directly with SAMM50. (A) Endogenous p62 was immunoprecipitated (IP) from HeLa cells followed by immunoblotting with antibodies for mitochondrial proteins identified in mass spectrometric analysis of endogenous p62 immunoprecipitates presented in Fig. S1 A. (B) Subcellular fractions of HeLa cells immunoblotted with the indicated antibodies. CALR, calreticulin. (C) High-resolution live-cell imaging of HeLa cells expressing mCherry-p62 stained with MitoTracker. Arrows indicate p62-containing mitochondrial fragments. Scale bars, 20 µm. (D) High-resolution imaging of HeLa cells stably coexpressing MIC19-EGFP and mCherry-p62 with enlarged images shown above and below. Arrows indicate association of p62 with mitochondria. Scale bars, 10 µm. (E) In vitro translated and [³⁵S]-methionine–labeled Myc-p62 tested in GST-pulldown experiments for interaction with selected mitochondrial proteins. Bound p62 was visualized by autoradiography (AR), and immobilized GST or GST-tagged proteins were stained with CBB. (F) Quantitative analysis of GST pulldowns in E, based on three independent experiments using Science Lab Image Gauge software (Fujifilm). Values are mean ± SD. **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (G) HeLa cell extracts immunoprecipitated with antibody to endogenous SAMM50 and immunoblotted with antibodies to indicated proteins. (H) GST pulldowns of in vitro translated Myc-SAMM50 and indicated GST-tagged proteins as in E. (I) Quantitative analysis of three independent GST pulldowns in H. Statistical values are mean ± SD. ***, P < 0.001; **, P < 0.005; *, P < 0.01; one-way ANOVA.