Phosphorylated pi3k

Immunoblot and immunohistochemical analyses were performed according to the method described previously [18 (link), 34 (link)]. Rabbit polyclonal antibodies against DTX3L/Dtx3l (Santa Cruz), phosphorylated threonine 202 in ERK1 and phosphorylated tyrosine 204 in ERK2 (Cell Signaling), phosphorylated tyrosine 397 in FAK (Invitrogen), phosphorylated MEK1/2 (Cell Signaling), PI3K and phosphorylated PI3K (Cell Signaling); rabbit monoclonal antibodies against Akt and phosphorylated Akt (Cell Signaling); and mouse monoclonal antibodies against alpha-TUBULIN (SIGMA), MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and FAK (Millipore) were used as first antibodies. Immunohistochemistry was performed according to the method previously described [34 (link)].

MTT powder (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan), rapamycin, METH powder, TRIZOL reagent, and RIPA buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). Staining reagents for AnnexinV and Caspase3/7 were obtained from Essen Bio (Ann Arbor, MI, USA). AnnexinV/PI apoptosis assay kit was purchased from BD Biosciences (San Diego, CA, USA). Antibodies against C/EBPβ, PI3K, Akt, mTOR, phosphorylated PI3K, phosphorylated Akt, Beclin1, LC3, Caspase3, Caspase7, and Bax were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-CHOP, anti-phosphorylated mTOR, anti-Bcl2, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against ATF6 were obtained from Abcam (Cambridge, UK). SYBR Premix Ex Taq was purchased from Takara (Shiga, Japan). The RT PreMix kit was obtained from Enzynomics (Daejeon, Korea). ECL Western blotting detection reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Proteins were extracted from the heart tissue in a 1x RIPA buffer (Melford, Ipswich, UK). The protein concentration was determined with Bradford assay. Total protein homogenates 20 μg/30 μl were denatured, separated on sodium dodecyl sulfate polyacrylamide electrophoresis gels, and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 2.5 % BSA in Tris- Buffered Saline Tween 20 for 1 h, before being incubated overnight at 4 °C with CD36 (1:1000, Cell Signalling Technology, Cambridge, UK), GLUT-4 (1:100, Santa Cruz, Biotechnology, Heidelberg, Germany), insulin receptor-β (1:1000, Cell Signalling Technology, Cambridge, UK), phosphorylated-PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), AKT, or phosphorylated-AKT (1:500, Abcam, Cambridge, UK). After washing the blots to remove excessive primary antibody binding, the blots were incubated for 1 h with a horseradish peroxydase conjugated secondary antibody at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), housekeeping protein, was used for loading control and protein normalization. The immunoreactive protein bands were developed using the Enhanced Chemiluminescence system (PerkinElmer, Rodgau-Juegesheim, Germany). The intensity of the immunoblot bands was detected with Chemismart 5100.