O response to surgical manipulation. Buprenorphine (0.1 mg/kg, sc) was administered

O response to surgical manipulation. Buprenorphine (0.1 mg/kg, sc) was administered for post-operative analgesia. Four weeks after the operation, the hearts, lungs, and tibiae of the mice were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) (mg/g), HW/ tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios of the different groups.Quantitative real-time RT-PCRReal-time PCR was performed to detect the mRNA expression levels of hypertrophic and fibrotic markers. Total RNA was extracted from frozen pulverized mouse cardiac tissue using TRIzol (Invitrogen, 15596-026). The yields and purities were spectrophotometrically estimated using the A260/A280 and A230/260 ratios, as measured with a SmartSpec Plus Spectrophotometer (Bio-Rad). The RNA (2 mg of each sample) was reverse-transcribed into cDNA using oligo(DT) primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). The PCR CAL 120 web amplifications were quantified using the LightCycler 480 SYBR Green 1 DprE1-IN-2 cost Master Mix (Roche,04707516001). The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.Protein 25331948 extraction and western blotting analysesThe left ventricles were harvested for western blotting analyses. They were first lysed in RIPA lysis buffer, and the protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23227) and an ELISA Reader(Synergy HT, Bio-Tek). The cell lysates (50 mg) were loaded into each lane and subjected to SDS-PAGE, and the proteins were then transferred onto Immobilon-FL transfer membranes (Millipore, IPFL00010). The membranes were incubated overnight at 4uC with primary antibodies against one of the following proteins: p-MEK1/2 (Cat#, 9154), T-MEK1/2 (Cat#,9122), p-ERK1/2 (Cat#,4370), T-ERK1/2 (Cat#,4695), p-P38 (Cat#,4511), T-P38 (Cat#,9212), p-JNK(Cat#,4668),T-JNK (Cat#,9258), p-PI3K(Cat#, 4228), TPI3K (Cat#,4257), p-AKT (Cat#,4060), T-AKT (Cat#,4691), PGSK3b (Cat#,9322), T-GSK3b (Cat#,9315), p-mTOR (Cat#,2971), T-mTOR (Cat#,2983), P-FOXO3A (Cat#,9465), T-FOXO3A (Cat#,2497), P-FOXO1 (Cat#,9461), T-FOXO1 (Cat#,2880), p-NFkB (Cat#,3033), T-NFkB (Cat#,4764), Bax (Cat#,2772), Bcl2(Cat#, 2870),Cleaved Caspase3(Cat#,9661),GAPDH(Cat#,2118),and IKKi(Cat#,3416). All antibodies were purchased from Cell Signaling Technology.The membranes were then incubated with goat anti-rabbit IgG (LICOR, 926-32211) for one hour IgG. The blots were scanned using a two-color infrared imaging system (Odyssey, LI-COR). SpecificEchocardiography and hemodynamic analysesEchocardiography was performed on anesthetized (1.5 isoflurane) mice using a MyLab 30CV (ESAOTE S. P. A) with a 15 MHz linear array ultrasound transducer. The left ventricle (LV) dimensions were assessed in the parasternal short-axis view. End-systole and end-diastole were defined as the phases that were associated with the smallest and largest areas of the LV, respectively. The end-systolic (LVESD) and end-diastolic (LVEDD) LV internal diameters and posterior wall end-diastolic thickness (PWT) were measured from the LV M-mode tracing with a sweep speed of 50 mm/s at the mid-papillary muscle level. The percentage of fractional shortening (FS) was calculated as (LVEDD-LVESD)/LVEDD6100. To perform the invasive hemodynamic measurements, we anesthetized the mice with 1.5 isoflurane, and a microtip transducer catheter (SPR-839, Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery and ad.O response to surgical manipulation. Buprenorphine (0.1 mg/kg, sc) was administered for post-operative analgesia. Four weeks after the operation, the hearts, lungs, and tibiae of the mice were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) (mg/g), HW/ tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios of the different groups.Quantitative real-time RT-PCRReal-time PCR was performed to detect the mRNA expression levels of hypertrophic and fibrotic markers. Total RNA was extracted from frozen pulverized mouse cardiac tissue using TRIzol (Invitrogen, 15596-026). The yields and purities were spectrophotometrically estimated using the A260/A280 and A230/260 ratios, as measured with a SmartSpec Plus Spectrophotometer (Bio-Rad). The RNA (2 mg of each sample) was reverse-transcribed into cDNA using oligo(DT) primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). The PCR amplifications were quantified using the LightCycler 480 SYBR Green 1 Master Mix (Roche,04707516001). The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.Protein 25331948 extraction and western blotting analysesThe left ventricles were harvested for western blotting analyses. They were first lysed in RIPA lysis buffer, and the protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23227) and an ELISA Reader(Synergy HT, Bio-Tek). The cell lysates (50 mg) were loaded into each lane and subjected to SDS-PAGE, and the proteins were then transferred onto Immobilon-FL transfer membranes (Millipore, IPFL00010). The membranes were incubated overnight at 4uC with primary antibodies against one of the following proteins: p-MEK1/2 (Cat#, 9154), T-MEK1/2 (Cat#,9122), p-ERK1/2 (Cat#,4370), T-ERK1/2 (Cat#,4695), p-P38 (Cat#,4511), T-P38 (Cat#,9212), p-JNK(Cat#,4668),T-JNK (Cat#,9258), p-PI3K(Cat#, 4228), TPI3K (Cat#,4257), p-AKT (Cat#,4060), T-AKT (Cat#,4691), PGSK3b (Cat#,9322), T-GSK3b (Cat#,9315), p-mTOR (Cat#,2971), T-mTOR (Cat#,2983), P-FOXO3A (Cat#,9465), T-FOXO3A (Cat#,2497), P-FOXO1 (Cat#,9461), T-FOXO1 (Cat#,2880), p-NFkB (Cat#,3033), T-NFkB (Cat#,4764), Bax (Cat#,2772), Bcl2(Cat#, 2870),Cleaved Caspase3(Cat#,9661),GAPDH(Cat#,2118),and IKKi(Cat#,3416). All antibodies were purchased from Cell Signaling Technology.The membranes were then incubated with goat anti-rabbit IgG (LICOR, 926-32211) for one hour IgG. The blots were scanned using a two-color infrared imaging system (Odyssey, LI-COR). SpecificEchocardiography and hemodynamic analysesEchocardiography was performed on anesthetized (1.5 isoflurane) mice using a MyLab 30CV (ESAOTE S. P. A) with a 15 MHz linear array ultrasound transducer. The left ventricle (LV) dimensions were assessed in the parasternal short-axis view. End-systole and end-diastole were defined as the phases that were associated with the smallest and largest areas of the LV, respectively. The end-systolic (LVESD) and end-diastolic (LVEDD) LV internal diameters and posterior wall end-diastolic thickness (PWT) were measured from the LV M-mode tracing with a sweep speed of 50 mm/s at the mid-papillary muscle level. The percentage of fractional shortening (FS) was calculated as (LVEDD-LVESD)/LVEDD6100. To perform the invasive hemodynamic measurements, we anesthetized the mice with 1.5 isoflurane, and a microtip transducer catheter (SPR-839, Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery and ad.