Smifh2

The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S). Mouse antibodies against the following antigens were purchased from Santa Cruz–Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942). Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals—cytochalasin D (5μM), CK869 (10μM), Dynasore (30μM), GSK269962 (1μM), Nocodazole (3μM), ML-141 (5μM), EHT 1864 (5μM), BMS-5 (1μM) and (-) blebbistatin (5μM); (2) Cell Signaling—LY294002 (10μM); (3) Abcam—SMIFH2 (12.5μM to 100μM).

To visualize the actin cytoskeleton, spreading cells were fixed and stained for immunohistochemistry according to standard procedures. Cells were fixed in 4% PFA in cytoskeleton buffer (10 mM PIPES, 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 1 mM MgCl₂) for 20 min on ice. Cells were then washed three times and permeabilized with 0.1% Triton X-100 solution in PBS for 10 min on ice and again washed three times. Cells were then blocked with 10% BSA in PBS (PBS-BSA) for a minimum of 1 h at 4 °C. To visualize the actin cytoskeleton, cells were stained with fluorescently labelled Phalloidin (conjugated with either Alexa 647, 555 or 405 fluorescent dyes). Cells were then imaged using a spinning-disk confocal microscope using a ×100 magnification objective. Cell peripheries were determined by staining with CellMask deep red plasma membrane stain (Invitrogen). For measurements investigating the effects of cytoskeletal inhibitors on cell spreading, cells were introduced into medium containing the inhibitor and allowed to spread on the GFP-coated surface (CK666 (100 µM), latrunculin B (5 µM), SMIFH2 (100 µM), blebbistatin (50 µM) inhibitors were purchased from Abcam). Cells were then fixed and stained according to the above procedure before being imaged using the Zeiss 980 Airyscan microscope and a ×40 water-immersion objective (Zen Blue).