Anti phospho akt ser473 antibody

CD3⁺ T cells and CD19⁺ B cells were separated from PBMCs using the IMag™ Cell Separation System (BD Biosciences, San Jose, CA, USA). The separated cells were then subjected to immunoblot analysis using the following antibodies: anti-AKT antibody (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-AKT (Ser473) antibody (Cell Signaling Technology), and ß-actin (SIGMA-ALDRICH, Saint Louis, MO, USA).

Rb2 (purity >98.0%) was obtained from Shanghai Yuanye Biotech Co. Ltd. (Shanghai, MO, China). Foetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Laboratory (Gaithersburg, MD), while dexamethasone and isobutylmethylxanthine (IBMX) were purchased from Sigma (St. Louis, MO, USA). Recombinant murine TNF-α was obtained from PeproTech(New Jersey, USA). Antibodies including rabbit anti-GAPDH (#2128), anti-apoptosis-associated speck-like protein (ASC) (#67,824), anti-IκBα (#AF5002) and anti-phospho-IκBα (#AF2002) antibodies were purchased from Affinity (Danvers, MA, USA). Antibodies against NLRP3 (#ab210491) and GSDMD (#ab209845) were obtained from Abcam (Cambridge, UK). A mouse anti-caspase-1 antibody (sc-514) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The following items were purchased from Cell Signalling Technology (Danvers, MA, USA): an anti-AKT antibody (#4691) and an anti-phospho-AKT (Ser473) antibody (#4060).

To characterize the in vitro insulin-like bioactivity, Akt phosphorylation, was applied as a measurement of insulin signaling pathway activation. H4IIE cells were treated with DMEM w/0.1% bovine serum albumin (BSA; Sigma) for 18 h for serum starvation. The serum-deprived cells were then treated with human insulin (Sigma), ExpressTec-ProINS-Tf, HEK-ProINS-Tf, or DMEM w/0.1% BSA. At the specific time points, cells were lysed with cell extraction buffer (Invitrogen) supplemented with protease inhibitor cocktail (Sigma) and phenylmethanesulfonyl fluoride (Sigma). The cell lysates were subjected to Western blot analysis using anti-phospho-Akt (Ser 473) antibody (#4060, Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibody (#5174, Cell Signaling Technology, Danvers, MA, USA). The Western blots were developed using Amersham ECL Plus kits (GE Health care, Piscataway, NJ, USA), and quantified using Quantity One 1-D Analysis software (Bio-Rad, Hercules, CA, USA).

Cells were lysed on ice with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail set III (Millipore, Billerica, MA). Total proteins were separated by electrophoresis using Any kD precast polyacrylamide gel (Bio-Rad, Hercules, CA), and transferred onto PVDF membrane. After blocking with 5% skim milk (Thermo Scientific) in TBST buffer, membranes were incubated with the first antibody, respectively: anti-MELK monoclonal antibody (in-house, previously described [8 (link)]), anti-β-actin antibody, anti-p21 antibody, anti-FOXO1 antibody, anti-FOXO3 antibody, anti-pan-AKT antibody, anti-phospho-AKT (Thr308) antibody, anti-phospho-AKT (Ser473) antibody (Cell Signaling, Danvers, MA), and anti-p53 antibody (Sigma-Aldrich). β-actin was used as a loading control.

ARVM and HL-1 cells were treated as indicated and lysed in buffer containing 50 mM HEPES (pH 7.4) (PromoCell, Heidelberg, Germany), 1 % Triton X-100 (Sigma-Aldrich, Munich, Germany), PhosSTOP and CompleteTM protease inhibitor cocktail (Roche, Basel, Switzerland). After incubation for 2 h at 4 °C, the suspension was centrifuged at 10,000g for 15 min. 5 μg protein sample of the total cell lysate was separated by SDS/PAGE (10 % gel) and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in tris-buffered saline (TBS) containing 0.1 % tween 20 and 5 % (w/v) non-fat dried skimmed milk powder and incubated overnight with anti-phospho Akt(Ser⁴⁷³) antibody, anti-phospho Akt(Thr³⁰⁸), anti-GAPDH antibody (all Cell Signalling Technology, Danvers, MA, USA) or anti-tubulin antibody (Abcam, Cambridge, UK). After washing, membranes were incubated with appropriate horseradish peroxidase-coupled secondary antibody and processed for enhanced chemiluminescence (ECL) detection using Immobilion horse radish peroxidase (HRP) substrate (Millipore, Darmstadt, Germany). Signals were visualised and evaluated on a VersaDoc 4000 MP Bio-Rad Laboratories work station and analysed by Quantity One analysis software (version 4.6.7) (both Bio-Rad Laboratories, Hercules, CA, USA).

Cell extracts were prepared, and Western blotting analysis was conducted as previously described.17 Nuclear extracts were prepared using a Nuclear Protein Extraction Kit (cat. no. R0050, Solarbio). The antibodies used for Western blotting were obtained from different resources: the anti‐Met antibody (cat. no. 8198), anti‐DYRK1A antibody (cat. no. 2771), anti‐phospho‐STAT3 (Tyr705) antibody (cat. no. 9145), anti‐cleaved PARP antibody (cat. no. 9541) and anti‐caspase‐3 antibody (cat. no. 9661) were purchased from Cell Signaling Technology; the anti‐phospho‐AKT (Ser473) antibody (cat. no. sc‐7985), anti‐Mcl‐1 antibody (cat. no. sc‐819), anti‐STAT3 antibody (cat. no. sc‐482), anti‐EGFR antibody (cat. no. sc‐373746) and anti‐GAPDH antibody (cat. no. sc‐25778) were obtained from Santa Cruz Biotechnology; the anti‐Lamin B (cat. no. 12987‐1‐AP) antibody was purchased from Proteintech Group.

PC12 cells were from Tongji Medical College, Huazhong Science and Technology University. LSPC was provided by Huazhong Agriculture University (China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan); anti-BDNF antibody was purchased from Elabscience (China); anti-CREB antibody, anti-phospho-CREB (Ser¹³³) antibody, anti-AKT antibody, anti-phospho-AKT (Ser⁴⁷³) antibody, anti-ERK1/2 antibody, anti-phospho-ERK1/2 (Thr202/Tyr²⁰⁴) antibody, and anti-GAPDH antibody were purchased from cell signaling; LY294002 inhibitor for PI3K and PD98059 inhibitor for ERK1/2 were purchased from Selleckchem; lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malonialdehyde (MDA) were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); gallic acid was purchased from DRE; procyanidin dimer B (PDB) was purchased from Fluka Co.; epigallocatechin gallate (ECG) was purchased from Chromadex; Annexin V-FITC for flow cytometry was purchased from BestBio; Hoechst staining for apoptosis analysis, BCA protein assay kit, and RIPA lysis solution was purchased from Beyotime; all other reagents were purchased from Sigma.