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. 2021 May 9;18(1):171–190. doi: 10.1080/15548627.2021.1922982

Figure 2.

Figure 2.

UBXN1 depends on PRKN for mitochondrial translocation, colocalizes and physically interacts with PRKN. (A) Hela cells transfected with expression plasmids for FLAG-UBXN1 and mitoYFP-T2A-MYC-PRKN or mitoYFP and treated with 25 µM CCCP for 6 h or left untreated as control. Cells were fixed and stained using mouse anti-FLAG antibodies and analyzed by confocal microscopy. Shown are representative images of three independent experiments. Scale bar: 20 µm. (B) Pearson’s correlation between FLAG-UBXN1 and mitoYFP was calculated. Box-plots show results of three independent experiments with at least 20 cells per condition. Statistical significance was evaluated using Student’s t-test. n.s. and *** denote p > 0.05 and p < 0.001, respectively. (C) Cells were transfected with expression plasmid for FLAG-UBXN1 and mCherry-PRKN, fixed and stained using anti-FLAG antibodies. The box plot depicts the ratio PRKNUBXN1:PRKNcytosolic of 28 cells from two independent experiments. Determination of the ratio PRKNUBXN1adjacent:PRKNcytosolic (UBXN1 adjacent: 10 pixel left or right and up or down) served as control. Statistical significance was evaluated using Student’s t-test with *** denoting p < 0.001. (D) HeLa cells were transfected with expression plasmids for YFP-PRKN or mCherry-PRKN and untagged UBXN1 and treated with 25 µM CCCP for 6 h or left untreated. Immunoprecipitation was performed using mouse anti-GFP antibodies. Whole cell lysates and precipitates were separated by SDS-PAGE and immunoblotted for PRKN and UBXN1 using rabbit anti-GFP and anti-UBXN1 antibodies, respectively.