Phospho akt ser473 d9e xp rabbit mab

Mouse corneal tissues or stromal cells were lysed using radioimmunoprecipitation lysis buffer to extract the proteins, and the total proteins were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, proteins were transferred to polyvinylidene fluoride membranes through Tris-glycine-SDS buffer and blocked. The main antibodies included Recombinant Anti-METTL3 (ab195352; Abcam), Anti-beta Actin (ab8227; Abcam), Akt Antibody (9272; Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (4060; Cell Signaling Technology), Recombinant Anti-PI 3 Kinase p85 alpha (ab191606; Abcam), Anti-PI 3 Kinase p85 alpha (phospho Y607) (ab182651; Abcam), Recombinant Anti-TNF alpha (ab183218; Abcam), Anti-IL-1 beta (ab9722; Abcam), and Anti-IL-6 (23431-1-AP; Proteintech). The membranes were incubated with the primary antibodies overnight. The primary antibody was recovered, and the membranes were rinsed three times with Tris-buffered saline with 0.1% Tween 20 and then incubated for 2 hours with a specific secondary antibody. Finally, the proteins were analyzed with enhanced chemiluminescence detection reagents for quantification (WBKLS0100; MilliporeSigma, Burlington, MA, USA).

BPN were isolated and characterized from the marine red alga Rhodomela confervoides in our laboratory as described previously.^{18 (link)} The compound used in the experiments was >95% purity as established by HPLC analysis. Palmitic acid (PA), insulin (INS), fatty acid-free BSA, 2,2-diphenyl-l-picrylhydrazyl (DPPH), streptozotocin (STZ) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-NBDG was obtained from Invitrogen (Carlsbad, CA, USA). Ni-NTA agarose resin was purchased from Qiagen (Hildon, Germany). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (Grand Island, NY, USA). IRS1 antibody, insulin receptor β (L55B10) mouse mAb, anti-phospho-IRβ (Tyr1146) rabbit IgG, phospho-Akt (Ser473) (D9E) XP rabbit mAb were obtained from Cell Signaling Technology (Danvers, MA, USA). p-IRS-1 Antibody (Tyr632)-R was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). GLUT1 antibody, anti-β-actin mouse monoclonal IgG and all secondary antibodies were obtained from Proteintech Group (Chicago, IL, USA). PVDF membrane for western blot analysis was purchased from Millipore (Bedford, MA, USA). The BCA Protein Assay Kit and Pierce™ ECL Western Blotting Substrate were purchased from Thermo Scientific (Waltham, MA, USA). ROS assay kit was obtained from Beyotime Biotechnology (Beyotime, Shanghai, China).

Phospho AKT (Ser473, D9E) XP rabbit mAb, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody, HER2/ErbB2 antibody, and AKT (pan; 40D4) mouse mAb were purchased from Cell Signaling Technology (Danvers, MA). ERK 1 (C-19) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX), and Anti-Pan-Ras (Ab-3) mouse mAb was from Sigma-Aldrich (St. Louis, MO). Anti-laminin 5 antibody and Alexa Fluor 568 goat anti-rabbit IgG were from Abcam (Cambridge, UK). Rictor mouse mAb 1G3P2C9 was obtained from Bethyl Laboratories (Montgomery, TX). Peroxidase AffiniPure goat anti-mouse IgG, peroxidase AffiniPure goat anti-rabbit IgG, and AMCA AffiniPure goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

D. latiflorus leaves were purchased from Maoshennongye Co., Ltd. (Shandong, China). HepG2 cell line was obtained from the National Infrastructure of Cell Line Resource, Peking Union Medical College (Beijing, China). Fetal bovine serum (FBS) Uruguay was purchased from Biowest (Nuaillé, France). Dulbecco's modified Eagle's medium (DMEM) with high glucose, liquid and other cell culture reagents were purchased from HyClone (Logan, UT, USA). AKT antibody, phospho-AKT (Ser473) (D9E) XP rabbit mAb, FOXO1 (C29H4) rabbit mAb, phospho-FOXO1 (Ser256) antibody, GSK-3β (D5C5Z) XP rabbit mAb, phospho-GSK-3β (Ser9) (D85E12) XP rabbit mAb, and GAPDH (14C10) rabbit mAb were purchased from Cell Signaling Technology (Beverly, MA, USA). The insulin growth factor 1 receptor (IGF1R) inhibitor GSK1904529A was purchased from Selleck Chemicals (Houston, TX, USA).