P70s6k

Western blot analysis was performed as described previously5 (link). Briefly, the placenta was homogenized to obtain protein samples. Then, the proteins (50 μg) were resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with anti-phosphorylation antibodies to Akt (1:1000, Cell Signaling Technology, Inc., USA), mTOR (1:1000, Cell Signaling), p70S6K (1:1000, Bioworld Technology), ERK1/2 (1:1000, Cell Signaling) and antibody of TNF-α (1:500, Bioworld Technology) at 4 °C overnight. The membranes were then incubated with an HRP-labeled secondary antibody and developed using an ECL detection kit (Millipore, MA, USA). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce) for 5 min, and then incubated with antibodies to Akt (1:1000, Cell Signaling), mTOR (1:1000, Cell Signaling), p70S6K (1:1000, Bioworld Technology), ERK1/2 (1:1000, Cell Signaling) and β-actin (1:1000, Cell Signaling). Western blot bands were scanned and analyzed with the National Institutes of Health Image image analysis software package.

Curcumin, Y15, 15d-PGJ2 and PD68235 were obtained from Sigma-Aldrich (St Louis, MO, USA). U0126 was obtained from Cell Signaling Technology (Danvers, MA, USA). Imatinib and fasudil were obtained from Nanjing EnoGene Biotechnology (Nanjing, China). Rapamycin was obtained from Xi'an Helin Biological Engineering (Xi'an, China). All these compounds were dissolved in dimethylsulfoxide (DMSO; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for experiments. Recombinant rat PDGF was obtained from Cell Sciences (Canton, MA, USA). Primary antibodies against VEGF, p-PI3K, PI3K, p-AKT and AKT were obtained from Nanjing EnoGene Biotechnology (Nanjing, China). Primary antibodies against α-smooth muscle actin (α-SMA), α(I) procollagen, fibronectin, p-PDGF-βR, PDGF-βR, p-FAK, FAK, GTP-RhoA and total-RhoA were obtained from Epitomics (San Francisco, CA, USA). Primary antibodies against PPAR-γ, p-ERK and ERK were obtained from Cell Signaling Technology. Primary antibodies against HIF-1α, VEGF-R2, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin were obtained from Bioworld Technology (Nanjing, China).

Adult male C57/BL6 mice were sacrificed at 8 weeks after TAC or sham surgery and heart samples were collected. Proteins were then extracted from these LV of hearts and assessed by western blotting analysis. The primary antibodies used were antibodies against P70/S6K, FGFR3, GAPDH (Bioworld Technology, Inc.), phosphor-P70/S6K and Caspase 3 (Cell Signaling Technology, Inc.), mTOR (Abcam, Inc), α-smooth muscle actin (α-SMA) (Abcam, Inc), atrial natriuretic peptide (ANP) (Abcam, Inc), SMARCD1 and SMARCA5 (ProteintechGroup, Inc.).