Phosphorylated gsk3β

Twenty four hours after surgery, proteins from different area of porcine myocardium were collected and diluted into 0.5 mg protein/mL for the measurement of pro-survival pathway proteins. Protein content was determined with BSA as a standard according to Bradford assay. Protein samples (20 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to PVDF membranes (Millipore, Billerica, MA) through electroblotting. Blots were probed with antibodies against phosphorylated Akt (Ser473), total Akt, phosphorylated GSK-3β (Ser9), total GSK-3β, phosphorylated ERK1/2 (pThr202/Tyr204) and total ERK1/2 (Cell Signaling, Boston, MA). The blots were then developed by enhanced chemiluminescence using SuperSignal west femto maximum sensitivity substrate (Pierce, Rockford, IL). Bio-Rad Image Lab™Version 3.0 software was used to calculate the numerical value of every blot.

Oridonin (purity ≥ 97%) was purchased from Xi’an Plant Bio-engineering Co., LTD (CAS: 28957–04-2, Xi’an, Shaanxi, China) and was analyzed and authenticated by high-performance liquid chromatography. 5-Fluorouracil (FU) and cisplatin were bought from Sigma-Aldrich (St. Louis, MO). Active AKT1 and AKT2 were purchased from SignalChem (Richmond, BC, Canada) and the GSK fusion protein for kinase assays was from Cell Signaling Technology (Beverly, MA). Antibodies to detect phosphorylated AKT, total AKT, phosphorylated GSK-3β, total GSK-3β, phosphorylated mTOR, total mTOR and cyclin B1 were also purchased from Cell Signaling Technology. The antibody to detect Bcl-2 was from Santa Cruz Biotechnology (Santa Cruz, CA) and the β-actin antibody was obtained from ZSGB-Bio Company (Beijing, China).

Using the same technique as for phosphorylated isoforms of GS, total GLUT4 (Pierce; 1:1000), total GSK3β D transduction Laboratories; 1:500), phosphorylated GSK3β (Cell Signalling; 1:1000) and AMPK (α1; 1:5000, α2; 1:15000 and β1; 1:1000, as described by Woods et al. [39 (link)], β2:1:3000, as described by Durante et al. [40 (link)] and pAMPK Thr172; 1:1000 from Cell Signalling) protein expression levels were measured in whole muscle homogenates. Antibodies directed against the AMPK γ1 and γ3 subunits did not detect the equine protein. All primary antibodies were diluted in 2% skimmed milk and incubated overnight at 4°C and all secondary antibodies were incubated for 45 minutes at room temperature. Appropriate HRP-conjugated secondary antibodies were used (Jackson IR; 0.16μg/ml for pGSK3β. Dako; 1:5000 for all others). GLUT4 and total GSK3β expression were quantified in muscle protein samples using α-actin (Sigma; 1:8000) as a loading control. phosphorylated GSK3β was expressed as a ratio to total GSK3β expression. AMPK expression was calculated and normalised according to a standard curve derived from protein homogenates of known quantities as is custom for this assay [41 (link)]. Chemiluminesence was measured using Luminata Forte HRP chemiluminescent substrate (Millipore) and quantified using ImageLab software (BioRad).