P pi3k

Cells were lysed in lysis buffer. After centrifugation at 12,000 g for 15 min at 4 °C, the supernatant was collected, and its protein concentration was determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). 30 μg proteins for each sample, were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluorid (PVDF) membranes (Millipore, Bedford, MA, USA), followed by being blocked with 5% bovine serum albumin in PBST (0.1% Tween-20 in PBS) for 1 h. The membranes were incubated with anti-p-Syk, p-PI3K, p-Akt, p-mTOR and β-actin (Cell signaling technology, Danvers, MA, USA) at 4 °C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were developed by enhance chemiluminscence detection regents (Pierce, Rockford, IL, USA).

50 mg of heart tissues removed from the border zones of infarct area were cut and homogenized in RIPA buffer (Cat# P0013B, Beyotime Biotechnology, Shanghai, China) added with protease inhibitor cocktail (Cat# 78430, Thermo Scientific, Waltham, MA, USA), and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and the protein concentration was quantified using a BCA protein assay (Cat# P0012, Beyotime Biotechnology, Shanghai, China). For western blotting experiments, protein samples were first separated with SurePAGE gels (Cat# M00656, GenScript, Nanjing, China) and transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA), which was incubated with one of the following primary antibodies in the indicated concentrations: CHI3L1 (1:500, #ab77528, Abcam, Cambridge, MA, USA), PAR2 (1:500; #YN2681, Immunoway Biotechnology, Plano, TX, USA), PI3K (1:1000, #4249, Cell Signaling Technology, Danvers, MA, USA), p-PI3K (1:500; #17366, Cell Signaling Technology, Danvers, MA, USA), ERK (1:1000, #4695, Cell Signaling Technology, Danvers, MA, USA), pERK (1:500, #4370, Cell Signaling Technology, Danvers, MA, USA), eNOS (1:800; #ab16798, Abcam, Cambridge, MA, USA), and GAPDH (1:3000, #TDY042, TDY biotech, Beijing, China).

Hepatocytes were harvested and washed with ice-cold PBS, then lysed with ice-cold RIPA lysis buffer (Beyotime Inst. Biotech, Beijing, China) with 1 mmol/L PMSF. Protein concentrations were calculated by BCA assay kits (Pierce, Rockford, IL, US). 20 μg of total cellular protein was subjected to 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Atlanta, GA, US). The membranes were blocked with 5% defatted milk powder at room temperature for 1 hr and then immunoblotting was performed with primary antibodies at 4°C overnight, followed by HRP-conjugated secondary antibody at room temperature for 1 h. Following each step, the membranes were washed five times with PBS-T (PBS-Twen 20) for 3 min. Finally, the blots were developed using the enhanced chemiluminescence (ECL) system. Antibodies for Beclin-1, LC3, caspase-3, -8, -9, Bid, p-Akt, Akt, PTEN, p-AMPK, AMPK, p-PI3K, PI3K, p-mTOR, mTOR and others were obtained from Cell Signaling Technology (Dancers, Mass, USA) and the dilution of all antibodies was 1:1000 according to the instructions of Cell Signaling Technology (Dancers, Mass, USA).

Equal amounts of protein were solubilized in sample buffer and electrophoresed in denaturing 10% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were saturated in TBST containing 5% BSA (Bio Sharp Sigma, A-4612) for an hour at room temperature and incubated with primary antibodies overnight at 4 °C. After incubation with a secondary antibody, the blots were then washed and detected with the ChemiDoc MP System (Bio-Rad Laboratories. Inc., Hercules, CA, USA). The antibodies used are as follows: GLI1 (Cell Signaling, 3538), p-AKT (Ser473) (Cell Signaling, 4060), AKT (Cell Signaling, 9272), p-PI3K (Cell Signaling, 17366), PI3K (Cell Signaling, 4257), GSK3α/β (Bioworld, BS1412), Cyclin D1 (Cell Signaling, 2978), CyclinD2 (Proteintech,10937-1-AP), CyclinD3 (Proteintech, 26755-1-AP), CDK4 (Proteintech, 11026-AP), CDK6 (Proteintech,19117-1-AP), GAPDH (Santa Cruz, CA, USA), and anti-rabbit IgG (Proteintech, SA00001-2).