Anti phosphor akt

Immunoblot analysis was performed as previously described 24 (link), 25 (link). The following antibodies were used: anti- FGFR2, from Santa Cruz Biotechnology, anti-GAPDH from Sigma-Aldrich, and anti-AKT, anti-phosphor-AKT, anti-ERK, anti-phosphor-ERK, anti-cyclinD1, anti-cyclinB1, and anti-phosphor-FGFR from Cell Signaling Technology. Secondary antibodies were from Sigma-Aldrich. Densitometry (Bio-Rad Laboratories) was applied to analyze the intensities of the respective protein bands.

Cells were harvested and lysed in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 3% sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM DL-dithiothreitol (DTT), and 0.1% bromophenol blue) with protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). The proteins (30 μg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Bovine serum albumin (5%) in TBS-T (1 mol/L Tris-HCl (pH 7.5), 0.8% NaCl and 0.1%Tween 20) was used to block the membrane. Then, the membrane was incubated with anti-Glut1 (Santa-Cruz, sc-7903, CA, USA), anti-LDHA (Santa-Cruz, sc-137243, CA, USA), anti-phosphor-S6K1 (Cell Signaling, #9209) and total S6K1 (Cell Signaling, #9202), anti-phosphor-AKT (Cell Signaling, #13038) and total Akt (Cell Signaling, #4691) and anti-β-actin (Sigma-Aldrich, A5441, St.Louis.USA) antibodies at 4°C overnight. The blots were then treated with an HRP-conjugated secondary antibody (Pierce)