Serine473 phosphorylated akt

HRMECs or tissues were lysed by lysis buffer (Cell Signaling, Topsfield, MA, USA). Total protein (50 μg) was loaded on Bio-Rad 4–20% gel system, transferred to PVDF membrane, and immunoblotted with one of the following primary antibodies: Serine473 phosphorylated Akt (Cat#4060, Cell Signaling), total Akt (Cat#4691,Cell Signaling), Threonine172 phosphorylated AMPK (Cat#2535, Cell Signaling), total AMPK (Cat#5832, Cell Signaling), Acetyl-CoA Carboxylase (Cat #3662, Cell Signaling), Phospho-Acetyl-CoA Carboxylase (Cat #11818, Cell Signaling), Claudin-1 (Cat#13995, Cell Signaling), zo-1 (Cat#13663, Cell Signaling), Claudin-5 (Cat#35-2500, Thermo-Fisher Scientific), Occludin (Cat#71-1500, Thermo-Fisher Scientific), and ß-Actin (Cat#SC-4778, Sigma). The secondary antibody was obtained from Cell Signaling Company. The membrane was exposed to Super-Signal Reagent (Pierce, IL, USA), and imaged on a Bio-Rad ChemiDoc Touch station (Bio-Rad).

Antibodies against tyrosine 627-phosphorylated Gab1 (corresponds to Y628 in murine Gab1, #3233), tyrosine 1068-phosphorylated EGFR (#3777), threonine 202- and tyrosine 204-phosphorylated ERK1/2 (#4370), tyrosine 705-phosphorylated STAT3 (#9131), serine 473-phosphorylated Akt (#4060), Akt (#4685), ERK1/2 (#4696), EGFR (#4267), STAT3 (#9139), vimentin (#5741), and E-cadherin (#3195) were obtained from Cell Signaling Technology (Danvers, MA, USA). The Gab1 specific antibody (#06–579) was purchased from Merck Millipore (Burlington, MA, USA). The α-tubulin (tubulin) specific antibody (#T5168) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
MEK inhibitor U0126, PI3K inhibitor LY294002 (both Cell Signaling Technology) and the EGFR inhibitor Gefitinib (Cayman Chemical Company, Ann Arbor, MI, USA) were dissolved in DMSO (Carl Roth, Karlsruhe, Germany). RPMI 1640 (phenol red-free) medium and penicillin/streptomycin were obtained from Life Technologies (Carlsbad, CA, USA). Fetal calf serum (FCS) was purchased from GE Healthcare (Chicago, IL, USA).