Migfilin does not regulate Stx7-SNAP29-Ykt6 complex assembly and SNAP29 O-GlcNAcylation. (A) KP4 cells were successfully overexpressed with migfilin. KP4 cells were overexpressed 3 × Flag(3f)-tagged migfilin. Immunoblotting analysis of migfilin levels in control (3f) and migfilin overexpressed (3f-Mig) KP4 cells under nutrient-rich (control), EBSS starvation (Starv.) condition. (B–D) Migfilin does not promote Stx7–SNAP29–Ykt6 complex assembly. (B) Control or migfilin knockdown cells were coexpressed 3 × Flag-tagged Stx7 (3F-Stx7), 3 × Flag-tagged Ykt6 (3F-Ykt6), 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 Stx7 and Ykt6, and GFP-SNAP29 or GFP in cell lysates was shown as input. Quantification of the level of 3 × Flag-tagged Ykt6 (C) or 3 × Flag-tagged Stx7 (D) precipitated by GFP-SNAP29 was shown. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 4 independent experiments. (E) OGT silencing promotes SNARE complex formation. Cell lysates (as specified in the figure) 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(3F)-tagged Vamp8, mCherry-Stx17, and GFP-SNAP29 or GFP in cell lysates was shown as input. (F) Depletion of migfilin does not increase SNAP29 O-GlcNAcylation. Control or migfilin knockdown cells (siMig #1) was overexpressed GFP-SNAP29. Cell lysates were immunoprecipitated (IPed) with anti-GFP nanobeads or control IgG (IgG) followed by immunoblotting with antibodies as indicated. The presence of target proteins in cell lysates was shown as input. Source data are available for this figure: SourceData FS4.