Figure 9.
SNARE complex reassembly rescues migfilin deficiency–induced autophagic defects. (A) Cell lysates (as specified in the figure) were extracted and used for immunoprecipitation with anti-GFP nano-beads. Immunoprecipitants were then analyzed by immunoblotting with antibodies as indicated. The presence of 3×Flag-tagged Vamp8, mCherry-Stx17, and GFP-SNAP29 in cell lysates was shown as input. (B) Quantification of the level of 3 × Flag-tagged Vamp8 precipitated by GFP-SNAP29. Data represent mean ± SE, *P < 0.05, **P < 0.01, one-way ANOVA. n = 3 independent experiments. (C) Immunoblotting analysis of LC3 and p62 protein levels in cells as indicated in the figure. (D and E) Quantification of LC3-II (D) and p62 (E) levels was shown. Data represent mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 4 independent experiments for each group. (F) KP4 cells were stably expressed with GFP-LC3. The number of GFP-LC3 puncta was measured. Representative images are shown in the left panel and quantification analysis is shown in the right panel. Scale bar: 5 µm (Magnify, 2 µm). At least 22 cells in each group were analyzed. Data represent mean ± SE, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. (G and H) KP4 cells were stably expressed with mRFP-GFP-LC3 reporter. Representative images of mRFP-LC3 puncta and GFP-LC3 puncta in cells as specified in the figure under control (G) or starvation (H) condition were shown. Quantification of the percentage of RFP+GFP+ puncta to total puncta was determined. Scale bar, 10 µm (Magnify, 2 µm). Data represent mean ± SE, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. At least 20 cells in each experiment were analyzed. (I) Cell lysates (as specified in the figure) were extracted and used for immunoprecipitation with anti-GFP nano-beads. Immunoprecipitants were then analyzed by immunoblotting with antibodies as indicated. The presence of 3×Flag-tagged Vamp8, Stx17 and GFP-SNAP29-WT or GFP-SNAP29-QM in cell lysates was shown as input. (J) Immunoblotting analysis of LC3 and p62 protein levels in cells as indicated in the figure. (K and L) Quantification of LC3-II (K) and p62 (L) levels was shown. Data represent mean ± SE, *P < 0.05, **P < 0.01, one-way ANOVA. n = 4 independent experiments for each group. (M) KP4 cells (as specified in the figure) were stably expressed with GFP-LC3. The number of GFP-LC3 puncta was measured. Representative images were shown in the left panel and quantification analysis was shown in the right panel. Scale bar: 10 µm (Magnify, 2 µm). At least 30 cells in each group were analyzed. Data represent mean ± SE, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. Source data are available for this figure: SourceData F9.

SNARE complex reassembly rescues migfilin deficiency–induced autophagic defects. (A) Cell lysates (as specified in the figure) were extracted and used for immunoprecipitation with anti-GFP nano-beads. Immunoprecipitants were then analyzed by immunoblotting with antibodies as indicated. The presence of 3×Flag-tagged Vamp8, mCherry-Stx17, and GFP-SNAP29 in cell lysates was shown as input. (B) Quantification of the level of 3 × Flag-tagged Vamp8 precipitated by GFP-SNAP29. Data represent mean ± SE, *P < 0.05, **P < 0.01, one-way ANOVA. n = 3 independent experiments. (C) Immunoblotting analysis of LC3 and p62 protein levels in cells as indicated in the figure. (D and E) Quantification of LC3-II (D) and p62 (E) levels was shown. Data represent mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 4 independent experiments for each group. (F) KP4 cells were stably expressed with GFP-LC3. The number of GFP-LC3 puncta was measured. Representative images are shown in the left panel and quantification analysis is shown in the right panel. Scale bar: 5 µm (Magnify, 2 µm). At least 22 cells in each group were analyzed. Data represent mean ± SE, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. (G and H) KP4 cells were stably expressed with mRFP-GFP-LC3 reporter. Representative images of mRFP-LC3 puncta and GFP-LC3 puncta in cells as specified in the figure under control (G) or starvation (H) condition were shown. Quantification of the percentage of RFP+GFP+ puncta to total puncta was determined. Scale bar, 10 µm (Magnify, 2 µm). Data represent mean ± SE, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. At least 20 cells in each experiment were analyzed. (I) Cell lysates (as specified in the figure) were extracted and used for immunoprecipitation with anti-GFP nano-beads. Immunoprecipitants were then analyzed by immunoblotting with antibodies as indicated. The presence of 3×Flag-tagged Vamp8, Stx17 and GFP-SNAP29-WT or GFP-SNAP29-QM in cell lysates was shown as input. (J) Immunoblotting analysis of LC3 and p62 protein levels in cells as indicated in the figure. (K and L) Quantification of LC3-II (K) and p62 (L) levels was shown. Data represent mean ± SE, *P < 0.05, **P < 0.01, one-way ANOVA. n = 4 independent experiments for each group. (M) KP4 cells (as specified in the figure) were stably expressed with GFP-LC3. The number of GFP-LC3 puncta was measured. Representative images were shown in the left panel and quantification analysis was shown in the right panel. Scale bar: 10 µm (Magnify, 2 µm). At least 30 cells in each group were analyzed. Data represent mean ± SE, **P < 0.01, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. Source data are available for this figure: SourceData F9.

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