N wasp

Mouse monoclonal antibodies against CHIKV (2D21-1), EEEV (1C2), VEEV (1A4A-1), WEEV (9F12), and RVFV envelope glycoprotein Gn (4D4) and nucleoprotein (R3-ID8-1-1) were obtained from US Army research Institute of Infectious Diseases (USAMRIID) archives [61 (link)]. Goat antibody against VEEV capsid (C) or envelope protein was generously provided by AlphaVax (via Kurt Kamrud). Rabbit antibodies against Arp3, actin, N-WASP, GAPDH, FLAG, and HA were obtained from Sigma-Aldrich. Mouse monoclonal antibodies against actin, CD44, GGA3, and Rac1 were purchased from BD Transduction Laboratories. Rabbit antibody against α/β-tubulin was obtained from Cell Signaling Technology. Sheep anti-human TGN46 antibody was from AbD Serotec. Alexa Fluor-conjugated antibodies and phalloidin, Hoechst 33342, and HCS CellMask Red were obtained from Life Technologies. All chemical inhibitors were purchased from Sigma-Aldrich, with the exception of EHT1864 (Tocris Bioscience). Cells were incubated with inhibitors for 1 h before addition of viruses unless otherwise indicated in the figure legends.

Western blotting was performed, as previously described (22 (link), 23 (link)). The following antibodies were used: Polyclonal rabbit antibodies for FLI1 (Cat. no. ab133485), ERK (ab184699) and FLI1ChIP grade (Cat. no. ab15289) were all purchased from Abcam; the WIP (Cat. no. PA5-51995) antibody was obtained from Invitrogen (Invitrogen; the phospho-ERK (Cat. no.9101S) antibody was obtained from Cell Signaling Technology (CST, Danvers, MA01923); the GAPDH (Cat. no. G9545) antibody was obtained from Sigma Aldrich; WASP (Cat. no. 10879-1-AP), N-WASP (Cat. no. 14306-1-AP), GATA1 (Cat. no. 10917-2-AP), β-actin antibodies (Cat. no. 20536 1 AP) were obtained from Proto Technology (Proteintech, Bucuresti, Romania); goat anti-mouse and goat anti-rabbit HRP conjugated antibodies were obtained from Cell Signaling Technology (Cat. nos. 5470s and 5151s, respectively). Antibody dilution was conducted according to the manufacturer’s instructions. The Odyssey system (LI COR Biosciences) and Bio were used to image proteins in western blot analysis.
The inhibitor of N-WASP (Wiskostatin) was obtained from Cayman Chemica (Cat. no. 15047-10). The development of our specific FLI1 inhibitory compounds A661 and A665 has previously been described (22 (link)).

Cells were plated onto sterile 13 mm glass coverslips that had been previously coated with 10 μgmL⁻¹ Rat tail Collagen I, 10 μgmL⁻¹ fibronectin, 10 μgmL⁻¹ laminin, 10 μgmL⁻¹ Poly-l-Lysine or 50% FBS diluted in sterile PBS. Cells were fixed with 4% paraformaldehyde for 10 min, RT. Coverslips were then washed three times with PBS before incubation with blocking buffer (0.05% Triton X-100, 5% BSA, PBS) for 15 min, rotating. Primary and secondary antibodies were diluted in blocking buffer and incubated with the coverslips in a dark, humidified chamber for 1 h. Coverslips were washed three times in PBS and once in MilliQ water before mounting with FluoromountG solution containing DAPI.
Imaging was conducted on either a Zeiss LSM 880 Airyscan confocal microscope with a heated incubator or a Nikon A1R confocal microscope with a heated stage incubator.
Antibodies used: WAVE2 (Santa Cruz: sc-33548), p34 ArpC2 (Millipore, Burlington, MA, USA; 07-227-1), Cortactin (Millipore; clone 4F11), α-Tubulin (Sigma; clone B512), phalloidin (ThermoFischer Scientific, Waltham, MA, USA), N-WASP (Sigma; HPA005750), Vinculin (Sigma; clone hVIN-1), Paxillin (BD Biosciences clone 349, Franklin Lakes, NJ, USA) and DAPI (Southern Biotech, Birmingham, AL, USA).