G box imager

Cells were lysed in lysis buffer (50mM Hepes, 150mM NaCl, 1mM EDTA, 1mM EGTA, 10% glycerol, 1% Triton X-100, 25mM NaF, 10μM ZnCl2) supplemented with 0,5mM phenylmethylsulfonyl fuoride (PMSF), 1mM orthovanadate, 1mM β-glycerophosphate and a protease inhibitor cocktail (Sigma-Aldrich, USA). Lysates were centrifuged at 13000 rpm for 10 min at 4°C. Pellets were discarded and protein concentration was adjusted using Bradford assay (BioRad). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, blocked 1h at room temperature in Tris-Buffered Saline / 5% non-fat dry milk / 0,1% Tween20, and blotted overnight with primary antibodies in blocking solution (rat monoclonal anti-PTK7 generated in the laboratory; mouse monoclonal αTubulin antibody, Sigma-Aldrich, USA). After extensive washings in TBS / 0,1% Tween20, filters were incubated 1h at room temperature (RT) with a HRP-conjugated secondary antibody before being revealed with an enhanced chemiluminescence substrate (West Pico, Thermo Scientific, USA). Acquisition was performed with a G-BOX imager (Ozyme, France).

Cells were lysed in lysis buffer (50 mm Hepes, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 25 mm NaF, and 10 μm ZnCl2) supplemented with 0.5 mm PMSF, 1 mm orthovanadate, 1 mm β-glycerophosphate, and a protease inhibitor mixture (Sigma-Aldrich). For immunoprecipitation, after preclearing with agarose beads and incubation with antibodies, protein G-agarose beads were added to the lysates, and bound immune complexes were recovered and washed three times in lysis buffer. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, blocked for 1 h at room temperature in Tris-buffered saline/5% nonfat dry milk/0.1% Tween 20, and blotted overnight with primary antibodies in blocking solution. After extensive washings in TBS/0.1% Tween 20, filters were incubated for 1 h at room temperature with an HRP-conjugated secondary antibody before being revealed with an enhanced chemiluminescence substrate (West Pico, Thermo Scientific). Acquisition was performed with a G-BOX imager (Ozyme).