Anti p akt ser473

Sorafenib was purchased from Sigma‐Aldrich (St. Louis, MO, USA). The compound A‐769662 and capsaicin were purchased from Tocris Bioscience (Bristol, UK). The primary antibodies anti‐CD133, anti‐ALDH1A1, anti‐pAkt‐ser473, p‐mTOR, pAMPKα1‐thr172, pACC‐ser79, anti‐cyclin D1, anti‐PGC1α, and anti‐PPARγ and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibody anti‐β‐catenin was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The primary antibody anti‐Hif‐1α was purchased from Novus (St. Louis, MO, USA). Anti‐alpha fetoprotein and peroxidase‐labelled secondary anti‐mouse IgG were purchased from Sigma‐Aldrich, and anti‐rabbit IgG was purchased from Calbiochem (San Diego, USA).

Western blot was conducted as previously described¹⁵ . Briefly, after removal of the brains, the bilateral hippocampal CA1 tissues were homogenized in ice-cold RIPA protein extraction buffer (Pulilai, China) containing 1% protease and phosphatase inhibitors. Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then electro-transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm). After blocking with 5% nonfat milk or 5% BSA, target proteins were detected using the following primary antibodies: anti-APP (1:20000, EPITOMICS), anti-growth associated protein-43 (GAP-43, 1:50000, EPITOMICS), anti-synaptophysin (SYN, 1:50000, EPITOMICS), anti-p-AKT (Ser473, 1:5000, Cell signaling), anti-AKT (1:20000, Cell signaling), anti-p-GSK-3β (Ser9, 1:1000, Cell signaling), anti-GSK-3β (1:5000, Abcam), anti-p-CRMP2 (Thr514, 1:100000, Abcam), anti-CRMP2 (1:50000, Abcam), and anti-GAPDH (1:20000, Neobioscience). Secondary antibodies were incubated for 1 h at 37 °C. The immunoreactive bands were detected using enhanced chemiluminescence detection kit (Millipore, USA) by exposure to X-ray films, and then analyzed using Image J software. Protein level was normalized to the matching densitometric value of the internal control GAPDH.

RIPA buffer (containing protease and phosphatase inhibitor cocktail, Sigma Aldrich, Saint-Quentin, France) was used for protein extraction. Proteins from each condition were subjected to SDS-PAGE and then transferred to nitrocellulose membranes, which were blocked with 5% BSA and incubated afterwards with primary antibodies against anti-Orai3 (1:300, Sigma Prestige, Saint Quentin, France), anti-SOX-2 (1:500, Sigma Aldrich), anti-Nanog (1:500, Cell Signaling, Saint Quentin, France), anti-Akt (1:1000, Cell signaling), anti-p-Akt (Ser473) (1:400, Cell Signaling). Tubulin antibody (1:2000, Sigma Aldrich) or GAPDH (1:4000, abcam, Cambridge, UK) were used as an internal control. Secondary antibodies, coupled to horseradish peroxidase, were then utilized. Bound antibodies were visualized using ECL chemiluminescent substrate (GE Healthcare, Saclay, France) and quantified using the densitometric analysis option in the Bio-Rad image acquisition system (Bio-Rad Laboratories, Marnes-la-Coquette, France). (Original blots are included in File S1 in the Supplementary Materials).

CK2α antisera was prepared as described^{44 (link)}, anti-p-Akt (Ser129) (catalog ab133458), anti-CK2β (catalog ab76025), anti-GLUT4 used for Wb (catalog ab62375) and anti-VAMP2 (catalog ab3347) antibodies were from Abcam (Cambridge, UK), anti-β-actin (catalog A5441) from Sigma-Aldrich. Antibodies raised against PTEN (catalog sc-7974), Akt1/2/3 (catalog sc-8312) Na/K ATPase (catalog sc-28800) and GLUT4 (catalog sc-1606) used for immunolocalization were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while anti-phospho-PTEN (Ser370) (catalog 07–889) was from Merck Millipore (Darmstadt, Germany). Anti-p-Akt (Thr308) (catalog #13038), anti-p-Akt (Ser473) (catalog #4060), anti-AS160 (catalog #2670), anti-p-AS160 (Thr642) (catalog #4288), anti-p-PRAS40 (Thr246) (catalog #13175), anti-GSK3β (Ser9) (catalog #5558), anti-p-FoxO1 (Ser253) (catalog #9461), anti-FoxO1 (catalog #2880) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

The N-fMLP peptide was synthesized and HPLC-purified by PRIMM (Milan, Italy). SDS-PAGE reagents were purchased from Bio-Rad (Hercules, CA, USA). Protein A/G Plus, anti-Flk1, anti-p-Flk1 (Tyr951), anti-p-Flk1 (Tyr996), anti-p-Flk1 (Tyr1175), anti-p-Flk1 (Tyr1214), anti-p-Tyr, anti-p47^phox, anti-p22^phox, anti-p-PLCγ1 (Y783), anti-PLCγ1, anti-PKCα, anti-PKCβII, anti-PKCζ, anti-PKCδ, anti-tubulin, anti-mouse, and anti-rabbit were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-PI3K (p85) and anti-p-Akt (Ser473) were from Cell Signaling Technology (Danvers, MA, USA). Anti-p-Ser, p22^phox siRNA (SI03078523), and scramble control siRNA (SI03650318) were from Qiagen (Hiden, Germany). FPR1 siRNA (L-005140-00) and scramble control (D-001810-10) were purchased from Dharmacon (Lafayette, CO, USA). Protein A-horseradish peroxidase was from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Pertussis toxin (PTX), apocynin, wortmannin, and LY294002 were from Sigma (St. Louis, MO, USA).