Figure 4.
Mapping of PEX14-interacting and pexophagy-inducing regions of OPTN. Flp-In T-REx 293 cells stably expressing po-mKeima were transfected with a plasmid encoding the indicated GFP-fusion protein and cultured in regular DMEM medium. After 2 days, the cells were processed for GFP-Trap and FACS analyses. 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, PEX14, 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 relative ratios (RR) of PEX14/GFP and PEX14/OPTN-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) Examples of po-mKeima–expressing cells displaying no, weak, moderate, or excessively high levels of pexophagy. The visuals depict overlay images of po-mKeima excited at 440 nm (false color: green) or 586 nm (false color: red). Yellowish (=low 586/440 excitation peak ratio) and reddish (=high 586/440 excitation peak ratio) dots represent peroxisomes and pexolysosomes, respectively (Li et al., 2023). Scale bar, 10 µm. (D) Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. (E) Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of three independent biological replicates. All conditions were statistically compared with the OPTN1–577-GFP condition (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). Source data are available for this figure: SourceData F4.

Mapping of PEX14-interacting and pexophagy-inducing regions of OPTN. Flp-In T-REx 293 cells stably expressing po-mKeima were transfected with a plasmid encoding the indicated GFP-fusion protein and cultured in regular DMEM medium. After 2 days, the cells were processed for GFP-Trap and FACS analyses. 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, PEX14, 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 relative ratios (RR) of PEX14/GFP and PEX14/OPTN-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) Examples of po-mKeima–expressing cells displaying no, weak, moderate, or excessively high levels of pexophagy. The visuals depict overlay images of po-mKeima excited at 440 nm (false color: green) or 586 nm (false color: red). Yellowish (=low 586/440 excitation peak ratio) and reddish (=high 586/440 excitation peak ratio) dots represent peroxisomes and pexolysosomes, respectively (Li et al., 2023). Scale bar, 10 µm. (D) Representative flow cytometry plots of each group (n = 3). The different colors represent the cell density at a given position. (E) Quantification of the percentage of cells in the gated area. The data are shown as the mean ± SD and represent the values of three independent biological replicates. All conditions were statistically compared with the OPTN1–577-GFP condition (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). Source data are available for this figure: SourceData F4.

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