Syntaxin 3

The mouse eyes were rapidly enucleated after cardiac perfusion and were incubated immediately in 4% paraformaldehyde for 30 min at room temperature. The eyes were equilibrated in 30% sucrose overnight followed by washing in PBS three times, and then embedded in optical cutting technology freezing medium and fast frozen. Sections were cut on Leica CM1950 cryostat (Leica, Germany) at 10 μm thickness and collected on the gelatin-coated slides.
Immunofluorescence assay was performed as previously described (Liu et al., 2015 (link)). The primary antibodies were as follows: MUNC18-1 (1:50, mouse monoclonal antibody, BD Biosciences, Canada), Syntaxin 3 (1:100, rabbit polyclonal antibody, Abcam, United Kingdom), FLAG (1:500, mouse monoclonal antibody, MBL, Japan).

Western blot, GST pull-down and co-immunoprecipitation assays were performed as previously described (Huang et al., 2015 (link)). Flag tagged human MUNC18-1, Flag tagged human syntaxin 1A (STX1A), Flag tagged human Syntaxin 3B (STX3B), and Flag tagged mouse Syntaxin 3B (Stx3B) were extracted from HeLa cells transfected with corresponding plasmids. GFP tagged human MUNC18-1 and GFP tagged worm UNC-64 were extracted from HeLa cells transfected with corresponding plasmids. GST tagged human MUNC18-1 was extracted from Escherichia coli Rosetta strain transformed with human MUNC18-1 cDNA plasmids. Mouse Syntaxin 3 (Stx3) was extracted from mouse retina lysate. The primary antibodies were as follows: MUNC18-1 (1:500, mouse monoclonal antibody, BD Biosciences, Canada), Syntaxin 3 (1:1000, rabbit polyclonal antibody, Abcam, United Kingdom), GFP (1:5000, rabbit polyclonal antibody, Proteintech, United States), FLAG (1:5000, mouse monoclonal antibody, MBL, Japan), GPADH (1:3000, mouse monoclonal antibody, Proteintech, United States), GST (1:5000, rabbit polyclonal antibody, ABclonal, United States), beta-actin (1:3000, mouse monoclonal antibody, CST, United States). Quantitative analysis of protein bands was performed by the Image J software¹.

Antibodies used in this study were ABCC2/ MRP2 (catalogue number MAB4150; Millipore, Burlington, MA), RAB11A (mouse) (catalogue number 610656; BioScience, Franklin Lakes, NJ), RAB11A (rabbit) (catalogue number ab128913; Abcam, Cambridge, MA), myosin Vb (catalogue number NBP1-87746; Novus), FLAG (catalogue number F3165; Sigma-Aldrich, St Louis, MO), UNC45A (rabbit) (catalogue number HPA039228; Merck, Kenilworth, NJ), UNC45A (mouse) (catalogue number ADI-SRA-1800-F; Enzo Life Sciences, Farmington, NY), beta-tubulin (catalogue number T4026; Merck), c-myc (catalogue number 631206; Takara, Kusatsu, Japan), ezrin (catalogue number SC58758; Santa Cruz Biotechnology, Dallas, TX), beta-actin (catalogue number A5441; Sigma-Aldrich), syntaxin-3 (catalogue number ab133750; Abcam), and munc18-2 (catalogue number ab103976; Abcam). Fluorescently labeled phalloidin (catalogue number A22284) was from Invitrogen.