Migfilin promotes SNAP29-Vamp8 complex assembly. (A) Control or migfilin knockdown cells were coexpressed 3×Flag-tagged Vamp8, mCherry-Stx17, and GFP-SNAP29 or GFP alone. Cell lysates were extracted and used for immunoprecipitation with anti-GFP nanobeads. Immunoprecipitants were then analyzed by immunoblotting with antibodies as indicated. The presence of 3×Flag-tagged Vamp8, mCherry-Stx17, and GFP-SNAP29 or GFP 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 < 0.01, unpaired two-tailed t test. n = 4 independent experiments. (C) Purified GST-tagged SNAP29 was used to pull down purified TrxHis-Vamp8 (8–73 aa) and TrxHis-Stx17 (142–225 aa) with or without purified MBP-tagged migfilin. The eluted proteins were analyzed by immunoblotting with the indicated antibodies. (D) Quantification of the level of TrxHis-Vamp8 (8–73 aa) precipitated by GST-SNAP29. Data represent mean ± SE, *P < 0.05, unpaired two-tailed t test. n = 3 independent experiments. (E) Control or migfilin knockdown cells were stably coexpressed GFP-SNAP29 and mCherry-Vamp8. Representative images showed that less GFP-SNAP29⁺ puncta colocalized with mCherry-Vamp8⁺LC3⁺ puncta in migfilin knockdown cells under starvation conditions. Scale bar, 10 µm (Magnify, 1 µm). Quantification of the percentage of GFP-SNAP29⁺ puncta to mCherry-Vamp8⁺LC3⁺ puncta was shown in the right panel. Data represent mean ± SE, **P < 0.01, unpaired two-tailed t test. n = 3 independent experiments with >37 cells for each group. (F) Control or migfilin knockdown cells were stably overexpressed with GFP-SNAP29. Representative images showed that less GFP-SNAP29⁺ puncta co-localized with lysosomes labeled with lysotracker red (Lyso-Red) in migfilin knockdown cells under starvation condition. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the percentage of the GFP-SNAP29⁺ puncta colocalizing with lysotracker red labeled-lysosomes was shown in the right panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with >22 cells for each group. (G) Direct interaction between migfilin and Vamp8. Purified MBP or MBP-tagged migfilin was used to pull down GST alone, GST-tagged Vamp8, or GST-tagged Stx17. The eluted proteins were analyzed by immunoblotting with the indicated antibodies. (H) KP4 were coexpressed 3 × Flag-tagged Vamp8 (3F-Vamp8). Cell lysates were IPed with anti-migfilin antibody or control IgG (mIgG) followed by immunoblotting with antibodies as indicated. The presence of target proteins in cell lysates was shown as input. (I) KP4 cells were co-expressed with GFP-Vamp8 and mCherry-Migfilin. The representative images showed that mCherry-Migfilin co-localized with GFP-Vamp8. Scale bar, 5 µm (Magnify, 2 µm). (J) KP4 cells were coexpressed with GFP-SNAP29 and 3 × Flag-tagged Vamp8. Representative images of in situ PLA analyses of SNAP29-Vamp8 interaction (red dots). Cell nuclei were visualized with DAPI (blue). Scale bar, 10 µm. Quantification of PLA puncta per cell was shown in the right panel. Data represent mean ± SE, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. (K) The co-localization ratio of mCherry-Migfilin with Lysotracker Green (Lyso-Green) in KP4 cells transfected with empty vector (vector) or 3 × Flag-tagged Vamp8 (Flag-Vamp8) was analyzed. 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 19 cells in each group were analyzed. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. Source data are available for this figure: SourceData F8.