Phosphatase protease inhibitors cocktail

Lysates of snap-frozen ccRCC cells were prepared in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5, 1% NP-40, 1 mM EDTA, 125 mM NaCl, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1× phosphatase/protease inhibitors cocktail; Roche). Cell lysates were centrifuged at 17,000 g for 20 min. A bicinchoninic acid assay (Bio-Rad Laboratories) was used to quantify total protein. 30 mg/lane of lysates mixed with 1× reducing Laemmli buffer (Bio-Rad Laboratories) was boiled at 95°C for 5 min, electrophoretically separated on SDS-PAGE gels, and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). Blots were probed with primary antibody to the respective proteins and afterward with HRP-conjugated secondary antibodies. Proteins were visualized with HRP substrate (Luminata Forte; EMD Millipore) on a camera system (ImageQuant LAS 4000; GE Healthcare).

Lysates of snap-frozen lung tissues (murine and human) and isolated murine pulmonary cells were prepared in RIPA buffer [50 mM TRIS (pH 7.5), 1% NP-40, 1 mM EDTA, 125 mM NaCl, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1× phosphatase/protease inhibitors cocktail (Roche Applied Science, Hercules, CA, USA)]. Cell lysates were centrifuged at 17 000 × g for 20 min. A bicinchoninic acid assay (Bio-Rad, Life Sciences Research, Hercules, CA, USA) was used to quantify total protein. Lysates (30 μg/lane) mixed with 1× reducing Laemmli buffer (Bio-Rad) were boiled at 95°C for 5 min, electrophoretically separated on SDS–PAGE gels, and transferred onto nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibody to the respective proteins and afterwards with HRP-conjugated secondary antibodies. Proteins were visualized with HRP substrate (Luminata Forte, Millipore, Madrid, Spain) on ImageQuant LAS 4000 (GE Healthcare Life Sciences, Madrid, Spain). Alternatively, human samples were blocked in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), incubated overnight at 4°C with primary fluorescent-labelled antibodies, and visualized on an Odyssey Imaging System (Licor). The intensity of the bands was quantified using ImageQuant 5.2 or ImageJ (NIH, Bethesda, MD, USA).