Fig 1: Torin1 induces PMP70 ubiquitination and degradation. (A) Representative fluorescence images of HeLa cells treated with Torin1 (1 µM) or DMSO for 24 h as indicated and immunostained for PMP70. (B) Quantification of peroxisome numbers (green puncta) in cells treated as in A. Values are mean ± SD, n = 3 independent experiments, calculated using >90 cells. (C) HeLa cells were treated with Torin1 for the indicated time. The cell lysates were immunoblotted with the indicated antibodies. (D) Quantification of the protein levels in C. (E) HeLa cells were transfected with HA-Ub for 24 h and then treated with Torin1 (1 µM) for 24 h and MG132 (10 µM) for 4 h. Cell lysates were then subjected to a ubiquitination assay and immunoprecipitated with anti-PMP70 antibody, followed by WB analysis. (F) HeLa cells were treated with DMSO, Torin1 (1 µM for 24 h), MG132 (10 µM for 4 h), Baf-A1 (40 nM for 24 h), and folimycin (10 nM for 24 h) as indicated and immunoblotted with PMP70, catalase, PEX19, PEX3, PEX13, and ß-actin antibodies. (G) Quantification of F. In D and G, the protein levels were quantified with ImageJ, then normalized to ß-actin. Values are mean ± SD, n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, P > 0.05 by two-tailed Student’s t test.
Supplier Page from Abcam for Anti-PEX13 antibody