Phospho akt p akts473

Cell lysis, protein extraction, and immunoblotting were performed as described^{83 (link)}. Protein concentrations were quantified using Bradford assays (Bio-Rad) with bovine serum albumin (BSA) as standard, and a SpectraMax M5 microplate reader. Samples were resolved by SDS-PAGE on 4–12% gradient polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and detected by immunoblotting. As primary antibodies, we used rabbit monoclonal antibodies to Smad2, phospho-Smad2 (Ser^465/467), Smad3, Akt, and phospho-Akt (p-Akt^S473) from Cell Signaling. Antibody against phospho-Smad3 (Ser^423/425) was from Abcam, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Sigma. For immunoblotting, immunoreactive bands were visualized using Western Lighting Plus ECL (Perkin Elmer).

HTO was obtained from Medalchemy (Alicante, Spain). The RPMI medium, triolein, RNaseA and propidium iodide were purchased from Sigma-Aldrich (St. Louis, USA), with penicillin-streptomycin purchased from Biowest (Nuaillé, France) and the fetal bovine serum (FBS) from Biosera (Boussens, France). The recombinant human EGF was obtained from R&D systems (Minneapolis, USA), while sodium orthovanadate was from Sigma-Aldrich (St. Louis, USA) and the protease inhibitors were from Roche (Roche, Basel, Switzerland). NBD-sphingomyelin and NBD-glucosylceramide were purchased from Larodan (Solna, Sweden), and NBD-ceramide from Invitrogen, Molecular Probes. The antibodies used to probe Western blots were raised against LC3BI-II, ERK, phospho-ERK (p-ERK), Akt and phospho-Akt (p-Akt S473), and they were purchased from Cell Signaling (Danvers, MA, USA). The antibody against α tubulin was purchased from Sigma-Aldrich (St. Louis, USA) and the antibodies against EGFR and p-EGFR (Y1068) were from Abcam (Cambridge, UK). The anti-EGFR antibody 930 used to stain the cell surface and to assess internalization was kindly provided by Genentech (San Francisco, USA).