Anti akt antibody 9272

Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN). Recombinant human CCL2 was obtained from BioLegend (San Diego, CA). Antibodies used in western blotting, IHC, immunocytochemistry, and flow cytometry were as follows: rabbit anti-human CCR2 antibody (ab21667), rabbit anti-human CCR4 antibody (ab83250), goat anti-human CCR4 antibody (ab1669), rabbit anti-goat immunoglobulin (Ig)G H&L (fluorescein isothiocyanate; FITC) antibody (6737), goat anti-rabbit IgG H&L (FITC) antibody (6717), anti-mannose receptor antibody (ab64693), and anti-CCR7 antibody (ab103404) obtained from Abcam (Cambridge, MA); anti-Akt antibody (9272), anti-phospho-Akt (Thr308) antibody (9275), anti-phospho-Akt (Ser473) antibody (9271), and anti-rabbit IgG HRP-linked antibody (7074) obtained from Cell Signaling Technology (Danvers, MA); and anti-GAPDH antibody (NB300-221) obtained from Novus Biologicals (Littleton, CO). The antagonists used in the neutralizing assay were a selective CCR2 antagonist (ab120812) obtained from Abcam and a CCR4 antagonist (SC-221406) obtained from Santa Cruz Biotechnology (Dallas, TX). The Akt inhibitor AZD5363 (S8019) was obtained from Selleckchem (Houston, TX).

Total protein lysates (20ng) of BAL samples or THP-1 transfected cells were lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitors for phosphoprotein and total protein analyses. Lysates were separated in 12% SDS-PAGE, transferred to 0.45nm nitrocellulose membrane (Biorad), followed by Western blot detection of pAKT^ser473, total AKT and b-actin, with anti-pAKT^ser473 antibody #9271, (Cell Signaling), anti-AKT antibody # 9272 (Cell Signaling) and anti-b-actin monoclonal antibody (Chemicon). Appropriate HRP conjugated secondary antibodies (Chemicon) were used and immunodetection was performed with enhanced chemiluminescence reagent Luminata^TM (Millipore). Bands were visualised with the ChemiDoc XRS+ system (Biorad) and densitometry analyses were performed using Image Lab ^TM software (Biorad).

Anti-pAkt (Ser473) antibody (#4051) and anti-Akt antibody (#9272) were purchased from Cell Signaling Technology (Beverly, MA, USA). LY294002 (PI3K inhibitor), MK-2206 (Akt inhibitor) and U0126 (MEK inhibitor) were purchased from Selleck Chemicals LLC (Houston, TX, USA). Insulin, IGF-1, dexamethasone, PA, TNF-α and IL-1β and metformin were purchased from Sigma Aldrich (St. Louis, MO, USA). Specific pathogen-free (SPF) male C57BL/6 mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Male obese diabetic db/db mice, with a C57BL/KsJ genetic background were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China) at 8 weeks of age. Animal were maintained and used according to the NIH Guide for the Care and Use of Laboratory Animals. All animal studies conducted were approved by Animal Research Committee of Tongji Medical College, HUST.