Nitrocellulose membrane

Total RIPA extracts were prepared from melanoma cells grown to 80% confluence in 6-well plates. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Hercules, CA, USA). Samples (50 to 80 µg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Courtaboeuf, France) using standard techniques. After 1 h at room temperature in blocking buffer (5% non-fat milk in PBS containing 0.1% Tween-20), nitrocellulose membranes were incubated overnight at 4 °C with rabbit anti-Patched antibody (Abcam ab53715; 1/1000, Cambridge, UK) or mouse anti-β-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako, Courtaboeuf, France). Detection was carried out with an ECL Prime Western Blotting detection reagent (Amersham, Courtaboeuf, France) on a Fusion FX imager (Vilber Lourmat, Collegien, France), and analyses were performed using ImageJ software.

H413 clone-1 cells were treated with different conditions as described above. Whole cell proteins were prepared by scraping the cells in cold PBS, extracted in SDS sample buffer, separated by SDS-PAGE using 5% to 12% gradient mini-gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked overnight with 3% BSA (Sigma) in 0.1 mol·L^-1 Tris buffered salts solution pH 7.4 (TBS). The nitrocellulose membranes were then incubated with rabbit polyclonal antibodies to human occludin (ab31721), JAM-A (ab106114), claudin-1 (ab15098), claudin-4 (ab53156), ZO-1 (ab59720), claudin-15 (ab215354; all 1 μg·mL⁻¹, Abcam, Cambridge, UK), and β-actin (0.1 μg·mL⁻¹, GenTex, Zeeland, MI, USA) in 0.05% Tween20/TBS for 4 h. β-actin was used as a loading control. After the incubation, the membranes were washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1 500 in Tween20/TBS for 2 h. Bound antibodies were displayed with AP substrate (Bio-Rad) after the development of reactivity for proteins.

The protein expression of ARFGEF1 in C33A cells and tissues was assessed by performing Western blotting. The Tissue or Cell Total Protein Extraction Kit (Sangon Biotech) was used to extract total proteins. Subsequently, the equivalent proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore, Plano, TX, USA). The nitrocellulose membranes were stained with rabbit anti-human ARFGEF1 (1:1000 dilution, Abcam, Cambridge, MA, USA) or GAPDH (1:1000 dilution, Abcam) antibodies, and then incubated with goat anti-rabbit horseradish peroxidase (HRP)-IgG (1:2000 dilution, Abcam) antibodies. The immunoreactive bands were visualized with electrochemiluminescence reagents (Millipore). Finally, the Image J software was used to analyze the densitometric readings of the immunoreactive bands [27 (link)].

Cell lysate proteins were resolved by SDS-PAGE using precast 4%–20% NuPAGE Bis–Tris Gels (Thermo Fisher Scientific, Waltham, MA) and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) using the standard immunoblotting technique. nitrocellulose membranes were processed with different specific primary antibodies, e.g., anti-HIF-1α (ab51608), anti-β actin (ab8227), anti-band 3 (ab108414), anti-phosphotyrosine (ab179530), and anti-phospho (Y359)–band 3 (ab77236) (Abcam, Cambridge, MA, USA). Appropriate HRP-conjugated goat anti-mouse IgG (ab97040) and anti-rabbit IgG (ab205718) secondary antibodies were also obtained from Abcam (Cambridge, MA).

Total RIPA extracts from cells or tumor homogenates were prepared. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Marnes-la-Coquette, France). Samples (50 to 80 µg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Bath, UK) using standard techniques. After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1% Tween-20, and 5% non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with rabbit anti-Patched antibody (Abcam ab53715; 1/1000), rabbit anti-Phospho-Erk1/2 antibody (Cell Signaling Technology (Leiden, The Netherlands); 1/1000), rabbit anti-Erk1/2 antibody (Cell Signaling Technology; 1/1000), or mouse anti-β-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako-Agilent, Santa Clara, CA, USA). Detection was carried out with an ECL Prime Western blotting detection reagent (Amersham) on a Fusion FX imager (Vilber Lourmat, Collegien, France), and analyses were performed using ImageJ software.

Total RIPA extracts from melanoma cells were prepared. Samples were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Amersham) using standard techniques. After 1 hr at room temperature (RT) in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1%Tween-20, and 4% non-fat milk), nitrocellulose membranes were incubated overnight at 4°C with rabbit anti-Patched antiserum (Ab39266 from Abcam 1/1000) and monoclonal mouse anti-βtubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated 45 min with anti-mouse (1:5000) or anti-rabbit (1:3000) immunoglobulin coupled to horseradish peroxidase (Dako). Detection was carried out with an ECL kit (Millipore) on a Las3000 (Fuji).