Migfilin associates with SNAP29. (A) Volcano plot showing migfilin (FBLIM1) binding proteins identified using migfilin immunoprecipitation (IP) followed by Mass Spectrometry (MS) from KP4 cells under starvation conditions. Red dots indicated migfilin (FBLIM1) and its significant interactor SNAP29. (B) KP4 (left panel) and SW1990 (right panel) cell lysates were immunoprecipitated (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. (C) KP4 (upper panel) and SW1990 (lower panel) cell lysates were IPed with anti-SNAP29 antibody or control IgG (rIgG) followed by immunoblotting with antibodies as indicated. The presence of target proteins in cell lysates was shown as input. (D) KP4 cells were coexpressed with GFP-SNAP29 and mCherry-Migfilin. The representative images showed that mCherry-Migfilin co-localized with GFP-SNAP29. Scale bar, 10 µm (Magnify, 2 µm). (E) Representative images of in situ PLA analyses of migfilin-SNAP29 interaction (red dots) in KP4 cells under both control and starvation condition. 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, unpaired two-tailed t test. n = 3 independent experiments. (F) Direct interaction between SNAP29 and migfilin. Purified MBP or MBP-tagged migfilin was used to pull down GST alone or GST-tagged SNAP29. The eluted proteins were analyzed by immunoblotting with the indicated antibodies.