Pi3k p85

Cryptotanshione (CPT) obtained from National Institutes for Food and Drug Control (Beijing, China) was prepared into a 10 mM stock solution dissolved by DMSO and stored at -20°C for later use. MTT (KeyGEN BioTECH, Jiangsu, China), DMEM-High Glucose medium (Hyclone company), Fetal bovine serum (Corning Cell Gro, Australia), 0.25% trypsin (Gibco, ThermoFisher Scinetific Inc, USA). Lipofectamine^TM 2000 Reagent was from Invitrogen Inc, USA. GPER specific agonist G-1 and antagonist G-15 were obtained from Cayman Chemical (Michigan, USA). The primary antibodies were listed below: cyclin D, cyclin B and cyclin A, Bax, Bcl-2, caspase-3, PI3K (p85) and GPER (Abcam, USA), p-AKT (Ser473, Cell Signaling Technology, USA). GPER siRNA, non-target siRNA and PI3K inhibitor LY294002 were purchased from Santa Cruz Biotechnology, USA.

Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from Gibco. Earle's balanced salt solution (EBSS) and propranolol were purchased from Sigma‐Aldrich. Rapamycin (RAPA), 3‐methyladenine (3‐MA) and chloroquine (CQ) were obtained from MedChemExpress, and ZVAD‐FMK, necrostatin‐1, liproxstatin‐1, SB203580, SP600125, SC79 and 740Y‐P were purchased from SelleckChem. Then, these reagents were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals or water and stored at −80°C. Primary antibodies against Akt, p‐Akt, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, Erk1/2 and p‐Erk1/2 were purchased from Cell Signalling Technology. Primary antibodies against alpha‐smooth muscle actin (α‐SMA), fibronectin (FN), LC3B, P62, p‐PI3K p85, PI3K p85, ATG9b, ATG9a, mTOR, p‐mTOR, ATG12, ATG5 and anti‐ubiquitin were procured from Abcam. Primary antibodies against beta‐actin, alpha‐tubulin, GAPDH and HRP‐conjugated secondary antibodies were obtained from Proteintech. DyLight 549‐conjugated and DyLight 488‐conjugated secondary antibodies were provided by Abbkine.

For the western blot analysis, hearts were lysed in RIPA buffer, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors and then centrifuged at 12,000 rpm for 15 min at 4°C. Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay (Beyotime, China). Aliquots of total heart extract (30–40 µg) were loaded onto SDS-PAGE gels of different concentrations and then transferred to PVDF membranes. The membranes were blocked in 5% nonfat dry milk solution in a triethanolamine-buffered saline with Tween 20 (TBST) and then incubated with commercial antibodies specific for Bax, Bcl-2, Beclin1, LC3, GAPDH (CST, CA, USA), p-mTOR, mTOR, p-AKT (s473), AKT1/2/3, and PI3K p85 (Abcam, CA, USA) at 4°C overnight. On the second day, the membranes were washed in TBST three times. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a 1 : 5000 dilution at room temperature for 1 h. After incubation in an ECL (Pierce Biosciences, USA) solution, membranes were exposed to Syngene Gene Genius. The intensity of protein bands was semiquantified using image analysis software.