P pdk1 ser241

Treated and control whole cell lysates were prepared in lysis buffer (Cell Signaling, Danvers, MA, USA). Eighty to one-hundred μg of protein were electrophoresed in a 10% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Proteins were electro-blotted onto PVDF membrane in a 50 mM Tris-base, 20% methanol, and 40 mM glycine electrophoresis buffer. Membranes were incubated in 5% non-fat dry milk in PBST (Phosphate 100 mM, KCl 27 mM, NaCl 1.37 M pH 7.4 after 1X dilution; 0.2% Tween-20) for 1 h. Blots were probed with primary antibody overnight at 4°C in 2% BSA in PBST, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) in 5% dry milk in PBST for 1 h at room temperature. Bound antibodies were detected by Super Signal West Pico Chemiluminescent Substrate detection reagent (Pierce/Thermo Scientific, Rockford, IL, USA) and visualized by autoradiography. The primary antibodies used for Western blot analysis were: anti-GLIPR1 (Novus Biologicals, Littleton, CO, USA), p-AKT Ser473 (Cell Signaling), p-GSK3beta Ser9 (Cell Signaling), p-PDK1 Ser241 (Cell Signaling), anti-CX3R1, GAPDH and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Gastric cancer cells were lysed with Cell Signaling lysis buffer (Cat#40–040, Millipore, Bellerica, USA). Protein concentration was determined via BCA analysis kit (ThermoScientific, Waltham, USA). For phospho-immunoblotting using p-ERK Thr202/Tyr204 (Cat#4376), p-AKT T308 (Cat#9275), p-PDK1 Ser241 (Cat#3438) approximately 50ug of protein was loaded, for total anti-ERK (Cat#9101), AKT (Cat#C67E7), PDK1 (Cat#3062), and actin (Cat#4967, all Cell Signaling, Danvers, USA) 5–10 µg, onto 4–20% SDS/Polyacrylamide gels. Proteins were transferred to nitrocellulose blotting paper via the dry HEP-OWL1 system (ThermoScientific, Waltham, MA). Bands were visualized via the Odyssey luminescence scanner (Li-Cor, Lincoln, USA).

Untreated as well as transfected whole cell lysates were prepared using the cOmplete Lysis-M kit (Roche Applied Science, Indianapolis, IN). Protein samples were prepared by combining lysates with NuPAGE Sample Reducing Agent, NuPAGE LDS Sample Buffer, and nuclease-free water (Life Technologies) and then boiling for 5 minutes at 100°C. Samples were loaded onto NuPAGE 4-12% Bis Tris Gels (Life Technologies), separated by SDS-PAGE, and then transferred to PVDF membranes. Proteins were blocked in 5% non-fat milk and incubated with the appropriate antibody in 5% BSA (Sigma-Aldrich). The following antibodies were used for Western blot analyses: AKT (#9272), pAKT Thr308 (#4056), pAKT Ser473 (#4058), CASP7 (#9492), Fox01 (#2880), pFox01 Ser256 (#9461), GSK-3β (#9315), pGSK-3β Ser9 (#9336), INNPL1 (#2839), p70 S6 Kinase (#2708), PDK1 (#5662), pPDK1 Ser241 (#3438), PI3K p85 (#4292), and GAPDH (#2118) [Cell Signaling, Danvers MA]; and pGSK-3β Tyr216 (#75745) [Abcam Inc., Cambridge, MA]. Protein bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL).