Lung cancer cells lines were lysed with a total lysis buffer (NaCl 150 mM, EDTA 1 mM, Hepes pH 7.5 50 mM, Triton X 1%, Glycerol 10%) supplemented with protease and phosphatase inhibitors cocktail. After the lysis, cells were centrifuged at 14000 rpm for 15 min. Protein concentration was evaluated using Bradford Reagent, 5x concentrate assay. Total protein extracts (30–50 μg) were boiled into Laemmli Sample buffer (2X) for 5 min and analyzed by Western blotting, using a 4–15% Mini-PROTEAN TGX Stain-Free precast gels. After electrophoresis, the proteins were transferred to a 0.45 nitrocellulose filters (GE Healthcare, Life Sciences #10600003). Immunoblots were then probed overnight at 4 °C with specific antibodies in DPBS-0,1% tween-1% BSA and proteins detection was performed by using appropriate peroxidase-conjugated secondary antibodies and chemiluminescence reagent (BIORAD, #170–5060). Immunoprecipitation was performed using whole-cell lysates. Proteins were extracted with lysis buffer containing 150 mM NaCl, 1 mM EDTA, 50 mM Hepes (pH 7.5), 1% Triton X-100 and 10% glycerol. Then lysates were incubated with antibodies overnight at 4 °C on a rotator. Protein A/G-PLUS-Agarose beads (Santa Cruz, #2003) were added and incubated for 2 h at 4 °C with rotation. Beads bound with immunoreactive complexes were washed four times with cold lysis buffer. Immunoprecipitated complexes were boiled for 5 min and subsequently analyzed with Western Blot.

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