In the second protocol, hearts were stabilized for 10 min and perfused for selleck 15 min, before being subjected to a wortman Inhibitors,Modulators,Libraries nin solution for 5 min pre ischaemia. After the 25 min total global ischaemic period, hearts were reper fused for 3 min with the wortmannin solution, before reverting to the drug free Krebs Henseleit buffer for the rest of the 27 min reperfusion period. Functional and bio chemical measurements were taken. Mechanical Function Parameters measured Functional measurements were taken during pre ischae mia and at 5 min, 10 min and 30 min into reperfusion. Heart rate and left ventricle developed pressure were measured. LVDevP was calcu lated as the difference between left ventricular systolic and diastolic pressures. The rate pressure product was Inhibitors,Modulators,Libraries calculated as the product of heart rate and LVDevP.
Biochemical Analysis To assess myocardial biochemical Inhibitors,Modulators,Libraries function, hearts, from all groups, were freeze clamped 10 min into reperfusion with Wollenberger clamps precooled in liquid nitrogen. Cardiac proteins were extracted with a lysis buffer contain ing 20 mM Tris. 20 mM p nitrophenylphosphate. 1 mM EGTA. 50 mM NaF. 0. 1 sodium orthovanadate. 1 mM phenylmethyl sulfonyl fluoride . 1 mM dithiothre itol . 10 gml aprotinin. The tissue lysates were diluted in Laemmli sample buffer, boiled for 5 min and 60g protein was separated by 10% PAGE SDS gel elec trophoresis. The lysate protein content was determined using the Bradford technique. The separated proteins were transferred to a PVDF membrane. These membranes were routinely stained with Ponceau Red for visualization of proteins.
Nonspesific binding sites on the membranses were blocked with Inhibitors,Modulators,Libraries 5% fat free milk in Tris buffered saline 0. 1% Tween 20 and then incubated with the primary antibodies that recognize PKBAkt and total PKB Akt, PI3 K, PDK1, FKHR, GSK 3 , cleaved caspase 3, cleaved PARP and PTEN. Membranes were subsequently washed with large volumes of TBST and the immobilized antibody conjugated with a diluted horse radish peroxidase labaled secondary antibody. After thorough washing with TBST, membranes were covered with ECL detection reagents and quickly exposed to an autoradiography film to detect light emission through a non radioactive method. Films were densitometrically analyzed and phoshorylated protein values were corrected for minor differences in protein loading, if required.
Inhibitors,Modulators,Libraries Antibod ies were purchased from Cell Signalling Technology and all other chemicals were obtained from Sigma. Data analysis Values are expressed as mean standard error of the mean. Some functional values are presented as percent age change from the baseline values. Results were com no pared by using a one way ANOVA with a Bonferoni Multiple Comparison as a post hoc test. P 0. 05 was con sidered as statistically significant. Abbreviations A beta. C control. cm centimeter. CO2 carbon dioxide.