Wave2

Lysates were collected on ice by scraping cells in RIPA buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1X protease and phosphatase inhibitors). The tubes were centrifuged for 10 min at 15,000 rpm and 4 °C. The lysate was transferred to a clean Eppendorf tube and measured using Precision Red.
The 40 μg of protein lysate was resolved on NuPAGE Novex 4–12% Bis-Tris gels and transferred onto a nitrocellulose membrane Bio-Rad system. Membranes were blocked with 5% BSA in TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 20 min prior to overnight incubation with the primary antibody at 4 °C on a shaking incubator. Membranes were then washed three times for 5 min each in TBS-T. Membranes were then incubated at room temperature for 1 hour with secondary AlexaFluor conjugated antibodies, after which the blots were washed again for 5 min in TBS-T three times before being imaged on the Li-Cor Odyssey CLx machine. Images were then analysed using the Image Studio Lite Version 5.2. Lysates were harvested on 4 independent occasions from NCKAP1^fl/fl and NCKAP1^-/- MEFs.
Antibodies used: NCKAP1 (Abcam, Cambridge, UK: ab126061), CYFIP1 (Abcam: ab156016), WAVE2 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-33548) and α-Tubulin (Sigma, St. Louis, MO, USA; clone B512) as the loading control.

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).