Anti calretinin

Nuclear and cytoplasmic protein fractions were prepared using the Nuclear Extraction KIT (#ab113474, Abcam) following the manufacturer’s instructions. Equal amounts (40 μg) were separated by 12.5% SDS-PAGE. Membranes were probed with anti-Calretinin (1:10,000; CR 7699/4), anti-FAK (1:1,000), rabbit monoclonal anti-Histone H3 (1:1,000; Cell Signaling Technology) and rabbit polyclonal anti-α-Tubulin (1:1,000; Cell Signaling Technology).

Immunohistochemistry of formalin-fixed, paraffin-embedded tissues from human explants was performed with the Histostain-Plus detection System according to the manufacturer's protocol (Life Technologies). Sections were dewaxed and rehydrated, and antigen retrieval was performed by heating the slides in 10 mM citric acid buffer (pH 6.0) for 15 min. Sections were incubated overnight at 4 °C in a humidified chamber with a 1 : 10 dilution of antibody to p16 (BD) and 1 : 50 NF2 (Santa Cruz). Control IHC experiments (data not shown) were performed without primary antibody. All sections were counterstained with Gill's haematoxylin and mounted for digital slide scanning.
For immunofluorescence, isolated cells were first fixed in 4% paraformaldehyde and where indicated permeabilized with 0.1% Triton in PBS-Tween (0.1% vol/vol), prior to blocking in 5% goat serum in PBS-Tween. Cells were then incubated with primary antibody 1 : 50 anti-Podoplanin (Santa Cruz) without prior permeabilization; 1 : 50 anti-Calretinin (Cell Signaling) overnight at 4 °C. Cells were washed three times for 10 min each with 1 × PBS, incubated for 1 h with secondary antibody (in blocking solution), washed three times with 1 × PBS, and counterstained with DAPI and mounted for confocal microscopy.

Protein extracts (cleared lysates) were obtained from cells grown to 70% confluence using standard RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (Quartett GmbH, Berlin, Germany) and 1 mM sodium orthovanadate (Na₃V0₄; Sigma). Cells were collected using a cell scraper, incubated on ice for 5-10 min and centrifuged at 12,000 x g for 20 min at 4°C. Proteins (40 μg) were separated by 10% SDS-PAGE, transferred onto nitrocellulose membranes [7 (link)], blocked with 5% BSA in TBS-Tween for 1 h and incubated overnight at 4°C with the following primary antibodies diluted in 2% BSA: rabbit polyclonal anti-Calretinin (1:10,000; CR 7699/4), rabbit polyclonal anti-FAK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-p-FAK Tyr³⁹⁷ (1:1,000; Cell Signaling Technology), and rabbit polyclonal anti-GAPDH (1:5,000; Sigma); followed by incubation with secondary goat anti-rabbit or anti-mouse (HRP)-labeled antibodies (Sigma) at a dilution of 1:10,000. The signals were detected as described in [7 (link)]. For the EMT analysis membranes were incubated overnight at 4°C with a rabbit monoclonal anti-E-cadherin antibody (1:1,000; #3195; Cell Signaling Technology) and a mouse monoclonal N-cadherin (1:1,000; # 610920; BD Bioscience).