Mapping of the OPTN-interacting region of PEX14 and assessment of its expression levels on OPTN-mediated pexophagy. Flp-In T-REx 293 cells stably expressing po-mKeima were (co-)transfected with (a) plasmid(s) encoding the indicated protein(s) and cultured in regular DMEM medium. After 2 days, the cells were processed for GFP-Trap and FACS analysis. Pexophagy was measured by gating single-cell GFP-positive populations for decreases in fluorescence intensity in the neutral channel. (A) Samples of protein extracts (input) and the GFP-Trap were processed for SDS-PAGE followed by IB using antibodies to GFP, OPTN, and GAPDH. Representative blots are shown. Specific protein bands are marked by arrows. Molecular mass markers (in kDa) are indicated on the left. (B) Densitometry quantifications of the RR of OPTN/GFP and OPTN/PEX14-GFP fusion proteins retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both normalized to 100%. Bars represent the mean ± SD of three biological replicates. (C and E) Representative flow cytometry plots of each group (n = 2–3). The different colors represent the cell density at a given position. (D and F) Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of two to three independent biological replicates. Relevant conditions were statistically compared (ns, nonsignificant; ***, P < 0.001). RR, relative ratio. Source data are available for this figure: SourceData F7.