Quantifying Proteins from Lung Tumors

Proteins from lung tumors of SPC/c-Myc-transgenic mice and/or non-transgenic animals were extracted and quantified as previously reported [7 (link)]. Seventy-five or 100 μg (50 μg in case of HEK293T over-expressing c-MYC) of total protein extracts were separated on 12.0% SDS-polyacrylamide gel and blotted onto PVDF membranes in 25 mM Tris and 190 mM glycine at 4 °C for 2 h at 350 mA or using the semi-dry iBlot transfer system (InVitrogen, Life Technologies). Specific antibodies were used in dilution given in parenthesis for the detection of anti-Traf4 (1:100), anti-hepsin (1:100), anti-Alk1 (= Acvrl1) (1:100), anti-Igfbp6 (1:100), anti-c-MYC (1:1000) and anti-α-Actinin (1:8000) purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany); anti-claudin2 (1:500) purchased from Zymed Laboratories, Inc. (San Francisco, CA, USA) and anti-Adam19 (1:1000) purchased from Cedarlane Laboratories (Burlington, Ontario, Canada). Antigen-antibody complexes were visualized using the ECL detection system NEN Life Science Products (PerkinElmer Life Science, Rodgau-Juegesheim, Germany) or ECL Select kit (GE-Healthcare, Milan, Italy) as recommended by the manufacturer and recorded with the Kodak IS 440 CF (Kodak, Biostep, Jahnsdorf, Germany) and a ChemiDoc XRS+ (BioRad, Milan, Italy).