Fig. 3

Fig. 3 PEX13 depletion increases membrane localization and ubiquitination of PEX5. (A) HeLa cells transfected with siCTRL, siPEX1, siPEX13, or siPEX14, fixed and stained for endogenous PEX5 (red) and ABCD3 (green). Prior to fixation cells were treated with 0.025% digitonin for 10 seconds to permeabilize the plasma membrane and washed with PBS. (B) Quantification of images in (A). Pearson’s correlation coefficient between PEX5 and ABCD3 was calculated (n = 3; 30 cells/trial). Error bars represent standard deviation. (C) Quantification of PEX5 on peroxisomes (A). Normalized mean fluorescent intensity of PEX5 within ABCD3 structures was calculated (n = 3; 30 cells/trial). Error bars represent standard deviation. (D) Flp-In T-Rex HEK293 cells stably expressing HA-tagged ubiquitin were transfected with siCTRL, siPEX13 or siPEX14 and treated with HCQ (10 μM) and doxycycline (1 μM) for 24 h. 3 h prior to lysis cells were treated with MG132 (10 μM). Immunoprecipitation (IP) with an antibody against the HA epitope tag was performed. Lysates and IP samples were separated by SDS-PAGE and probed for PEX5. (E) Immunofluorescent images of HeLa cells transfected with siCTRL or siPEX13 and co-transfected with USP30-Flag, fixed and stained for ABCD3 (red) and Flag (green). (F) Graph of ABCD3 density from images in (E) (n = 3; 30 cells/trial). Error bars represent standard deviation. (G) Immunoprecipitation of HA-ubiquitin in cells treated with the indicated siRNA as in D, however IP samples were treated with or without 5 mM BME and assessed on non-reducing SDS-PAGE. Full immunoblot of anti-PEX5 is presented in Figure S3B. Scale bars: 25 μm (A), 20 μm (F). Asterisks denote p-values; *p < 0.05, **p < 0.01.