P akt ser473

BCa cells were lysed for Western blotting and proteins were detected using the antibodies, GAPDH (Santa Cruz 32233), P-Ser473 AKT (Cell Signaling 4060), INPP4B (Santa Cruz 12318), Total AKT (Cell Signaling 9272), ERα (produced in our lab, SC1-1), and p27 (Santa Cruz 528). Tissue fixation and sectioning were processed as previously described [36 (link), 73 (link)]. Tissue sections were incubated with ERα antibodies (Santa Cruz, MC-20), P-Ser473 AKT (Cell Signaling 4060), INPP4B (Abcam EPR3108), Ki67 (Novocastra).

Total protein extraction from EDL and pgWAT was performed as previously described (Milkiewicz et al., 2011 (link)). The following polyclonal primary antibodies were used: Ser473-pAkt, Akt, Ser563-pHSL, HSL and α/β-tubulin (Cell Signaling Technology, #4058, #9272, #4139, #4107 and #2148, respectively) and β-actin (sc-47778, Santa Cruz Biotechnology). Secondary antibodies were goat anti-rabbit or anti-mouse IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories, # 111-035-003, 115-035-003, respectively). Membranes were developed using enhanced chemiluminescence (SuperSignal^TM Westpico, #34080, ThermoFisher Scientific) and densitometry analysis was performed with ImageJ Analysis Software (NIH).

The following antibodies were used for immunoblotting: PLCδ1, PLCδ3 (lab-made); β-actin, Tubulin (SIGMA); PKCα (BD Biosciences, San Jose, CA, USA); PKCβ₁, PKCθ, Caveolin1, Bax (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); and PKCɛ, ^Thr538p-PKCθ, Bad, Bcl-2, Akt, ^Ser473p-Akt, ERK, p-ERK, cleaved Caspase 9, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology Inc., Danver, MA, USA).

The immortalized human hepatic stellate cell line LX-2 was purchased from Xiang Ya Central Experiment Laboratory (China) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) in a humidified incubator at 37°C with 5% CO₂. SEA of S. japonicum was obtained from Jiangsu Institute of Parasitic Diseases (China). Primary antibodies for mouse double minute protein 2 (Mdm2), total-Akt (T-Akt) and p53 were purchased from Santa Cruz Biotechnology (USA). Primary antibodies for caspase-3 and phospho-Akt (Ser⁴⁷³, p-Akt) were purchased from Cell Signaling Technology (USA). The primary antibody for glyceraldehyde phosphate dehydrogenase (GAPDH) was provided by Goodhere (China). All of the secondary antibodies were obtained from Santa Cruz Biotechnology (USA). In addition, GW501516 (Santa Cruz, USA), which is a potent PPARβ/δ agonist, was also used to activate Akt signaling [15] (link).

The myotubes were incubated for 20 min in DMEM-low glucose-Glutamax in the presence or absence of 100 nM insulin. Afterwards, the cells were harvested in a RIPA buffer (Sigma, St. Louis, MO, USA) complemented with 10 µL/mL protease inhibitor, 10 µL/mL phosphatase I inhibitor and 10 µL/mL phosphatase II inhibitor. Afterwards, the protein concentration was determined by BCA reagent (Thermo Scientific, Rockfold, IL, USA). 20 µg of total protein were run on 4–15% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA), electroblotted onto PVDF membranes (Millipore, Bradford, MA, USA), and immunodetected with ChemiDoc MP imaging system (BioRad, Hercules, CA, USA) while using the following primary antibodies: GLUT4 (Santa Cruz Biotech, CA, USA), Ser9 pGSK-3β, Thr172pAMPK, Ser473 pAkt (Cell Signaling Technology, Danvers, MA, USA), Tyr-989 pIRS-1 (Abcam, Cambridge, UK), and Thr642 pAS160 (Gene Tex, CA, USA). Histone H3 (Cell Signalling Technology, Danvers, MA, USA) served as an internal control, with the exception of pAkt, where the internal control was GAPDH (Cell Signalling Technology, Danvers, MA, USA). The bound antibodies were visualized by an ECL system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and quantified using Chemi-Doc MP imaging system (Bio-Rad, Hercules, CA, USA).

Total proteins were isolated from 50 mg of whole hind limb muscles using T‐PER tissue protein extraction reagents (78510; Thermo Fisher Scientific, Inc.). Protein concentration was measured using the BCA protein assay kit (Pierce 23225; Thermo Fisher Scientific, Inc.). Total proteins (20 μg) were separated on Bolt 4–12% Bis‐Tris PlusGels (NW04125BOX; Thermo Fisher Scientific, Inc.) by electrophoresis, and the fractionated proteins were subsequently transferred to a nitrocellulose membrane using iBlot Gel Transfer Stacks Nitrocellulose (IB23001; Thermo Fisher Scientific, Inc.). The blots were probed using the following antibodies: AMP‐activated protein kinase α (AMPKα; SC‐74461; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Thr¹⁷²‐phosphorylated (p)‐AMPKα (sc‐33524, Santa Cruz Biotechnology, Inc.), AKT (no. 4691; Cell Signaling Technology, Inc., Danvers, MA, USA), Ser⁴⁷³‐p‐AKT (#4060; Cell Signaling Technology, Inc.), glucose transporter 4 (GLUT4; ab64; Abcam, cambridge, England) and β‐actin (sc‐477778; Santa Cruz Biotechnology, Inc.). The density of protein bands was analyzed using Bio‐Rad imaging software (Bio‐Rad Laboratories, Hercules, CA, USA). The individual values were originally expressed as a ratio of a standard (β‐actin content) and then expressed as a fold change of the control group value.