Anti tks5

Cells were grown on cover slips in dishes. Following treatment, the cells were fixed with 4% paraformaldehyde (in PBS) for 15 min. The fixed cells were blocked with 1% BSA (in PBS) containing 0.1% Triton X-100 for 30 min at room temperature. The cells were incubated with the indicated primary antibodies at 4 °C overnight. Alexa Fluor^® 568 Phalloidin was obtained from Invitrogen and anti-Tks5 was purchased from Santa Cruz Biotechnology. Following an overnight incubation, the cells were incubated with the corresponding fluorescent-conjugated secondary antibodies for 2 h at room temperature. DAPI (0.5 μg/ml; Vector Laboratories, Burlingame, CA, USA) was used to counterstain the nuclei. The images were obtained using a Leica TCP-SP8 confocal microscope (Leica, Wetzlar, Germany) and Zeiss ApoTome microscope (Carl Zeiss, Jena, Germany). The co-localization rate and Pearson’s co-localization coefficients were determined with LAS AF Image software (Leica). Co-localization rate is the value that indicates the extent of co-localization in percentage and is calculated as follows: 100 × (Co-localization Area/Area Foreground), where area foreground is the difference between total area of the image and area of the image background. The values of Pearson’s coefficients ranging from 0.5 to 1 are considered positive co-localization.

HUVEC cells were lysed in RIPA buffer (1% Triton X-100, 0.5% deoxy-cholate and 0.1% SDS) containing protease inhibitors Aprotinin, Leupeptin, Pepstatin, Bestatin (Sigma, 20 μg/ml each) and 1 mM PMSF. Lysates (10 μg) were subjected to SDS-PAGE on 8% gels then transferred to polyvinylidine difluoride membranes (Immobilon-P, Millipore). Membranes were blocked with 3% BSA (Sigma), incubated with the indicated primary antibodies then horseradish peroxidase-conjugated secondary antibodies (Amersham), and developed using enhanced chemiluminescence (ECL West Pico, Pierce). Primary antibodies used for western blotting in this study were: anti-beta actin (Sigma, A1978), anti-Tks4 (1:5000 dilution, SH3PXD2B-specific antibody^{20 (link)}) anti-Tks5 (1:200 dilution, Santa Cruz, sc-30122, generous gift by Dr. Buday, Biological Research Center, Hungarian Academy of Sciences).

Immunoblots were performed as described previously [9 (link)]. 50–80 μg protein lysate were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with the following primary antibodies: anti-α-Tubulin (Sigma, T6199; 1:5000), anti-β-Actin (Sigma, A5441; 1:5000), anti-HA (Roche, 3F10; 1:1000), anti-MLC2 (Cell Signaling Technology, 3672; 1:1000), anti-MYLK (Abcam, EP1458Y; 1:1000), anti-pMLC2 (Cell Signaling Technology, 3674; 1:1000), anti-pPYK2 (Cell Signaling Technology, 3291; 1:1000), anti-TKS5 (Santa Cruz, sc-30122; 1:1000) and anti-ZEB1 (Sigma Prestige, HPA027524; 1:5000).