Effect of verteporfin-induced high-MW p62 on its association with polyubiquitinated proteins and LC3.

(A) MCF-7 EGFP-LC3 or (B) BxPC-3 cells were exposed for 4 h to 0.1% DMSO or 10 µM verteporfin in complete medium. p62 was immunoprecipitated and the bound polyubiquitinated proteins were detected using an anti-(Ub)_n antibody. Immunoprecipitation was confirmed by western blot for p62. (C) Densitometry analysis was performed on the images presented in A and B using Quantity One software. (D) Using the same lysates prepared in (A) and (B), EGFP-LC3 was immunoprecipitated and bound p62 was detected in the IP fraction using an anti-GFP antibody. Immunoprecipitation was confirmed by western blot for GFP. Images presented for MCF-7 EGFP-LC3 cells are representative of at least 3 independent experiments. Densitometry was done using images from 2 of those experiments (mean ± S.D., n = 2) where the image quality was suitable for quantification. Images presented for BxPC-3 cells are representative of 2 independent experiments and densitometry was done using images from 1 experiment where the image quality was suitable for quantification.