Hpa005724

Cells were fixed with 4% PFA, washed 3 x with 0.2% Dulbecco/BSA and permeabilized with 0.1% Triton X-100 in TBS. Immunofluorescence stainings were performed as in (Tojkander et al., 2011 (link)). Images were acquired with a charge-coupled device camera (AxioCam HRm; Zeiss) on a microscope (Axio Imager.M2; Zeiss). AxioVision Rel. 4.8 (Zeiss) and PlanApo 63x/1.40 (oil) objective (Zeiss) was used for the image acquirement. The following reagents and antibodies were used for the stainings: Alexa phalloidin 488, 568, 594 and 647 (1:200–400 dilutions) (Life Technologies^TM), anti-cofilin-1 antibody (Abcam, ab11062), anti-VASP antibodies (1:50–100) (Sigma, HPA005724 and Enzo, IE273), VASP-phospho-T278 antibody (1:50) (ImmunoWay), VASP-phospho-S239 antibody (1:50) (Millipore, 16C2), anti-vinculin antibody (1:50) (Sigma, hVin-1). DAPI and secondary antibodies, which were conjugated to Alexa Fluor 488, Alexa Fluor 568/594, or Cy5 were from Life Technologies.

Cells were washed with cold PBS, scraped, and lysed in PBS, 1% Triton X-100 (with 0.3 mM PMSF and protease and phosphatase inhibitor cocktail (Pierce). Protein concentrations were measured using Bradford reagent (Sigma-Aldrich). Alternatively, cells were lysed after washes into 4x LSB-DTT buffer for obtaining total cell lysates. Lysates were briefly sonicated prior to boiling. Mixture of 5% milk/BSA was used for blocking. Following antibodies were used for detection with dilutions recommended by the manufacturers: rabbit polyclonal anti-VASP antibodies (Sigma, HPA005724 and Enzo, IE273), VASP-phospho-T278 antibodies (ImmunoWay and ECM Biosciences, VP2781), VASP-phospho-S239 antibodies (Millipore, 16C2; Sigma SAB4504565; Abcam, 16C2), anti-GAPDH (Sigma, G8795). Appropriate HRP-linked secondary antibodies (Promega) and ECL reagent (AmershamTM, GE Healthcare) were applied for chemiluminescence detection of the blots. Quantity One 4.1.1 program (Bio-Rad) was used to quantify the band intensities of blots.