Anti cortactin

COS‐7 cells were treated as previously described (Chen et al., 2002). Briefly, COS‐7 cells were fixed with 4.0% formaldehyde in PBS at room temperature for 15 min and permeabilized with 0.3% Triton X‐100 in PBS. After blocking with 2% BSA, anti‐Kv1.2 (1:200 dilution; bought from Abcam, Cambridge, UK) and anti‐cortactin (1:200 dilution; Abcam, Cambridge, UK) was applied, and incubated at 4°C overnight. The cells were then washed three times with PBS, and incubated with FITC‐labelled anti‐mouse IgG (1:2000 dilution, Life technologies; Paisley, UK)) or TRITC labelled anti‐rabbit IgG (1:200 dilution, Life technologies; Paisley, UK) at RT (22 ± 0.5°C) for 1 h The slides were mounted in Vectorshield (Vector Laboratories, Peterborough, United Kingdom) mounting medium supplemented with DAPI staining, and registered using Leica confocal microscope imaging system (Leica Microsystems GmbH; Wetzlar, Germany). The co‐localization analysis was performed with ImageJ software.

FFPE sections from primary MCC tumors were purchased from Origene and analyzed as previously described (83 (link)). The primary antibodies were CK20 (Dako; dilution, 1:50), MCPyV LTA CM2B4 (Santa Cruz Biotechnology; dilution, 1:125), and anti-cortactin (Abcam; dilution, 1:250). An isotype-matched irrelevant antibody was used as a negative control on sections of tissues in parallel, and a rabbit polyclonal isotype control antibody (Abcam) was used to match the cortactin primary antibody. The sections were incubated with appropriate secondary antibodies labeled with different fluorochromes [Alexa Fluor 488 IgG2B and 633 IgG2A (Invitrogen) and IgG(H+L)-tetramethyl rhodamine isocyanate (TRITC) (Jackson ImmunoResearch)]. All slides were mounted with Immuno-Mount, and images were captured with a Zeiss LSM 510 confocal microscope.

Cells were plated onto poly-L-lysine coated dishes and cultured to 80% confluency. Cells were harvested in ice-cold RIPA lysis buffer (Teknova), supplemented with protease inhibitors (complete cocktail, Roche) and phosphatase inhibitors (Halt cocktail, Sigma-Aldrich). SDS-PAGE was performed with 20 µg protein per sample, transferred to a polyvinylidene difluoride membrane (Immobilon), blocked with 5% BSA/TBST for 3 h at room temperature and incubated with anti-β-actin (Santa Cruz Biotechnology, sc-47778; 1 to 500), anti-cortactin (Abcam, ab33333; 1 to 1000), anti-E-cadherin (BD Transduction Laboratories, 610181; 1 to 1000), anti-FAK (Santa Cruz Biotechnology, sc-271126, 1 to 100), anti-phospho-FAK (Tyr397) (Invitrogen, 44625 G, 1 to 100) anti-N-cadherin (BD Transduction Laboratories, 610920; 1 to 500) and anti-Tks5 (Millipore, MABT336; 1 to 500) antibodies diluted in 5% BSA/TBST overnight at 4 °C. The membranes were then incubated with HRP conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technologies; 1 to 5000) antibodies diluted in 5% non-fat milk/TBST for 1 h at room temperature and proteins were visualized using chemiluminescence detection reagents (WesternBright, Advansta) and blot scanner (C-DiGit, LI-COR).