Ser473

Biochemical analysis of bone marrow cells plated on fibrinogen was performed as described previously (4 (link)). Lysates containing equal amounts of proteins were subjected to immunoblotting with antibodies against total and phospho-Akt (Thr-308 and Ser-473, Cell Signaling).

Prior to euthanasia, mice were injected i.p. with a bolus of 10 U/kg insulin or with saline and after 10 minutes adipose tissue, liver, and muscle were harvested and homogenized on ice in RIPA buffer with protease/phosphatase inhibitors. To determine the activation of the insulin signaling pathway, insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and protein kinase B (Akt) phosphorylation and total protein expression were measured by western blotting using the following antibodies: pIR (Y972, Abcam, 1 : 500 dil); IRbeta (Cell Signalling, 1 : 1,000 dil); pAkt (Ser473, Cell Signalling, 1 : 1,000 dil); Akt (Cell Signalling, 1 : 1,000 dil); pIRS-1 (Y896, Invitrogen, 1 : 500 dil); IRS-1 (Cell Signalling, 1 : 500 dil). β-Actin (Santa Cruz, 1 : 2,000 dil) was used to normalize in some cases for protein loading. Activation of the same proteins was also measured by western blotting in isolated adipocytes obtained from epididymal adipose tissue following collagenase digestion. Adipocytes were analyzed in the basal state or following stimulation with 5 nM insulin for 30 minutes.

All the antibodies were from Cell Signaling Technology (Danvers, MA, USA): mTOR (cat #2983), phospho-mTOR (Ser2481, cat #2974), phospho-mTOR (Ser2448, cat #5536), Rictor (cat #9476), phospho-Rictor (Thr1135, cat #3806), Raptor (cat #2280), AKT (cat #4691), phospho-AKT (Ser473, cat #4060), phospho-AKT (Thr308, cat #13038), phospho-AKT (Thr450, cat #9267), GSK-3β (cat #9315), phospho-GSK-3β (Ser9, cat #9336), PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 (Thr37/46, cat #2855), S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K (Thr389, cat #9234), Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen (Carlsbad, CA, USA). Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich (St Louis, MO, USA). Protease and phosphatase inhibitor cocktails were from Calbiochem (San Diego, CA, USA). Cycle Test Plus kit was from BD Bioscience (East Rutherford, NJ, USA). Super Signal West Pico imaging system was from Thermo Scientific (Rockford, IL, USA).

At the indicated time (24 h), treated A375, VR1, VR1-SgSkp2 cells were collected to investigate levels of relevant cascade protein or apoptotic markers by Western blot analysis. The following antibodies from Cell Signaling were used: p-ERK1/2 (#9101), p44/42 MAPK (ERK1/2; #9102), p- AKT (Ser473; #9271), AKT (#9272), cleaved PARP (#9185), or GAPDH (#3683). Skp2 antibody was purchased from Santa Cruz (sc-74477).

Immunoblotting has been described previously (1 (link)). Primary antibodies used include: SIRPα (1:1,000; #13379; Cell Signaling), phospho-Shp1 (1:1,000; Tyr564; #8849; Cell Signaling), total Shp1 (1:1,000; #3759; Cell Signaling), phospho-Shp2 (1:1,000; Tyr580; #3703; Cell Signaling), total Shp2 (1:1,000; #3397; Cell Signaling), phospho-Lck (1:1,000; Y394; #ab201567; Abcam), total Lck (1:1,000; #2752; Cell Signaling), phospho-SYK (1:1,000; Tyr525/526; #2710; Cell Signaling), total SYK (1:1,000; #13198; Cell Signaling), phospho-AKT (1:1,000; Ser473, #5082; Cell Signaling), total AKT (1:1,000; #9272; Cell Signaling), GAPDH (1:5,000; #5174; Cell Signaling), and β-actin (1:5,000; sc47778; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Cell Signaling) and goat anti-mouse secondary antibodies (1:5,000; Sigma-Aldrich) were used for detection. SignalFire™ Plus ECL reagent (#12630S, Cell Signaling) was used for protein expression detection as per the manufactures protocol. Fusion SL (Analis Instruments) was used as per manufacturer instructions and chemiluminescent captured using Fusion SL (Analis Instruments). Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or β-actin or GAPDH using Image J software.

Whole cell lysates were prepared using an radio immunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 50 mM Tris HCl, 1 mM EDTA, 0.24% sodium deoxycholate,1% Igepal, pH 7.5) to protect samples from protein degradation. NaF (25 mM) and Na3VO4 (2 mM), Aprotinin, leupeptin, pepstatin, and phenylmethylsulfonylfluoride (PMSF) were added to the lysis buffer. Whole cell lysates (20 μg) were separated on 9% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with milk (5%, w/v) diluted in Tris-buffered saline containing Tween20 (TBS-T, 0.05%). Blots were then incubated overnight at 4°C with appropriate primary antibodies. Antibodies included those targeting Akt (#4685), pAkt (Ser-473, Cell Signaling, Danvers, MA), Cx43 (abcam, gja1,ab11370, MA) and tubulin (abcam, ab7291, MA). Following primary antibody incubation, blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody diluted at 1∶5,000 (Cell Signaling) at RT for 1 h. Chemiluminescence was detected with ECL plus (Amersham Biosciences, Piscataway, NJ) and densitometry was performed via NIH ImageJ software.

Immunofluorescence staining of cardiac cryosections was performed as described previously with slight modifications [6 (link), 15 (link)]. For the determination of cardiac fibrosis, Masson trichrome staining was performed on cardiac tissue slices (4 μm, short-axis orientation, mid-ventricular level) as described previously [15 (link)]. Alternatively, cardiac slices were incubated with an anti-collagen III antibody (ab7778) from Abcam overnight at 4 °C. Secondary antibody (Cy3 AffiniPure goat anti-rabbit immunoglobulin G (IgG);111–165-144) was incubated for 3 h at room temperature in the dark.
For immunofluorescence staining of AKT in the myocardium, iCM-Akt1/2KO or WT mice were starved for 4 h, and received intraperitoneally insulin (3 IU/kg) 15 min before organ harvesting. Wheat germ agglutinin (Lectin from triticum vulgaris FITC conjugate; # L4895, Sigma-Aldrich, St. Louis, MO, USA)-labelled 8 μm slices were stained with either pAkt1 (Ser473; Cell Signalling #9018) or pAkt2 (S474; Cell Signalling #8599) antibody. Secondary antibody (Cy3 AffiniPure goat anti-rabbit immunoglobulin G (IgG);111–165-144) was incubated for 3 h at room temperature in the dark. The slides were mounted with DAPI Fluoromount-G (SouthernBiotech). Analyses were carried out using a Keyence immunofluorescence microscope (BZ 9000) and ImageJ software [30 (link)].