Wave2 h110

Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). XB130 monoclonal antibody (1:2) was generated as previously described [8 (link)]. Rabbit anti-XB130 (1:250) and rabbit anti-Tks5 (SH3#1) (1:500) was from EMD Millipore (Merck, Darmstadt, Germany). Anti-GAPDH (1:10000) and Na⁺/K⁺ ATPase (1:20000) antibodies were from Abcam PLC (Cambridge, UK). Rabbit anti-Rac1 (ARC03) (1:250) and mouse anti-Cdc42 (BK034) (1:500) were from Cytoskeleton, Inc. (Denver, CO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:10000) or anti-rabbit secondary (1:10000) antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ, USA). pEGFP-XB130 and p-EGFP-vector were constructed as previously described [11 (link)]. mCherry-vector and mCherry-Tks5 were supplied by GeneCopoeia (Rockville, MD, USA). Myc-Tks5 SH3#5 W1108A was provided by S. Courtneidge.

Western blots were performed as previously described [16 (link)]. Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000). Blots were incubated with SuperSignal West Pico or Femto ECL reagents (Thermo Fisher, Waltham, MA, USA) and imaged using a PxiTouch imaging system (SynGene, Bengaluru, Karnataka, India).