Ram px

Cells were harvested directly into lysis buffer (150 mM NaCl, 1 % Nonidet P-40, 50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, protease inhibitor cocktail) and 20 μg of total protein in NuPAGE LDS Sample Buffer (Life Technologies, USA) loaded on 10 % polyacrylamide gels, separated and transferred onto nitrocellulose membranes in Mini-PROTEAN Electrophoresis System (Bio-Rad, USA) for 90 min applying 100 V in transfer buffer (240 mM Tris, 190 mM glycine, 20 % methanol). Membranes were blocked in 5 % non-fat milk in PBS with 0.1 % Tween and incubated overnight with primary antibodies at 4 °C. Primary antibodies used: anti-p63 clone SFI-6 (DCS Innovative Diagnostik-Systeme, Germany), anti-TAp63 clone TAp63-4.1 and anti-ΔNp63 clone ΔNp63-1.1 and polyclonal ΔNp63-44 [4 (link)] and anti-actin AC40 (Sigma-Aldrich, USA). Membranes were then incubated with appropriate HRP-conjugated secondary antibodies (RAM-Px, SWAR-Px, Dako, Denmark) diluted 1 : 1000 for 1 h at room temperature. Signal was detected using ECL reagent (Amersham Pharmacia Biotech, UK).

The cells were trypsinized, washed in PBS and resuspended in lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, and protease inhibitor cocktail). The protein concentration was measured using a Bradford protein assay, and 20 μg of total protein in the NuPAGE LDS Sample Buffer (Life Technologies, USA) was loaded onto 10% polyacrylamide gels, separated and transferred onto nitrocellulose membranes using the Mini-PROTEAN Electrophoresis System (Bio-Rad, USA) for 90 min, applying 100 V in the transfer buffer (240 mM Tris, 190 mM glycine, and 20% methanol). The membranes were blocked in 5% non-fat milk in PBS with 0.1% Tween and incubated overnight with primary antibodies at 4°C. Primary antibodies used: ΔNp63-1.1 (ΔNp63, Moravian Biotechnology, Czech Republic), DC-10 (cytokeratin 18, Exbio, Prague, Czech Republic), DO-1 (p53, Moravian Biotechnology, Czech Republic), NCL-L-EGFR (EGFR, Novocastra, UK), vimentin—Dako—Clone V9, E-cadherin—Cell Signaling - 24E10, N-cadherin—Cell Signaling—D4R1H, AC40 (actin, Sigma-Aldrich, USA). The membranes were subsequently incubated with HRP-conjugated secondary antibody (RAM-Px, Dako, Denmark) for 1 h at room temperature. The signal was detected using ECL reagent (Amersham Pharmacia Biotech, UK).