Gpr40

The human neuroblastoma cell line SK-N-MC was obtained from Korean Cell Line Bank (Seoul, Korea). The antibodies of p-Akt (Thr308), p-Akt (Ser473), Akt, mTOR, Caveolin-1, Flotillin-2, p-Tau (Ser396), Tau, p-NF-κB p65 (Ser536), NF-κB p65, Lamin A/C, CBP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies of p-mTOR (Ser2448), p-p70S6K1 (Thr389) and p70S6K1, were acquired from Cell Signaling Technology (Beverly, MA, USA). Aβ, BACE1, HIF-1α and GPR40 antibodies were obtained from Abcam (Cambridge, MA, USA). The C99 antibody was purchased from EMD Millipore (Darmstadt, Germany). Horse radish peroxidase (HRP)-conjugated IgG was obtained from Jackson Immunoresearch (West Groove, PA, USA). PA, BSA, GW9508, ionomycin, PF4708671, LY294002 and rapamycin were purchased from Sigma Chemical Company (St. Louis, MO, USA). The Akt inhibitor, GW1100 and SN50 used here were purchased from Calbiochem (La Jolla, CA, USA). siRNAs for GPR40, GPR120, APP, BACE1, HIF-1α and non-targeting were obtained from Dharmacon (Lafayette, CO, USA).

Cell lysis was performed using the RIPA (including 1% PMSF) protein extraction solution (Solarbio, Beijing, China). The protein concentrations were measured using the BCA protein concentration assay kit (Solarbio, Beijing, China). SDS–polyacrylamide gel electrophoresis (PAGE) was conducted to separate the extracted proteins with different molecular weights. The target proteins were immediately transferred to nitrocellulose membranes, which were then incubated overnight at 4℃ with antibodies against β-actin (42 kDa; ZSGB-BIO, Beijing, China), β-Tubulin (55 kDa; ZSGB-BIO, Beijing, China), GPR40 (40 kDa; Abcam, Cambridge, USA), GPR120 (40 kDa; Abcam, Cambridge, USA), KLF7 (25 kDa; Abcam, Cambridge, USA), CCL2 (40 kDa; Abcam, Cambridge, USA), CCR2 (35 kDa; San Ying Biotechnology, Wuhan, China), Ki67 (384 kDa; Abcam, Cambridge, USA), and MMP2 (72 kDa; Abcam, Cambridge, USA). Afterward, the membranes were incubated with the secondary antibody at room temperature for 2 h. It needs to be explained that, in order to reduce the mutual interference of polyclonal antibody incubation among multiple antibodies, we have tailored according to MW markers in this study, and each membrane containing different target proteins is incubated separately with corresponding antibodies.The protein bands were detected based on chemiluminescence (ThermoScientific, Waltham, America).

hUCB-MSCs were obtained from MEDIPOST Co. Ltd (Seoul, Korea). Fetal bovine serum was bought from BioWhittaker Inc. (Walkersville, MO, USA). AA, A23187, bisindolylmaleimide I, BrdU, Indomethacin, LY294002, mitomycin C, Nordihydroguaiaretic acid (NDGA), rapamycin, 1-Aminobenzotriazole (1-ABT), and SB203580 were aquired from the Sigma Chemical Company (St Louis, MO, USA). Phospho-Akt^ser473, phospho-Akt^thr308, Akt1/2/3, β-Actin, collagen1A, collagen3A, collagen5A, fibronectin, phospho-p38, p38, phospho-JNK, JNK, p-ERK1/2, ERK, lamin A/C, MMP-12, pan-cadherin, PKCα, PKCɛ, PKCθ, PKCζ, PKC, Phospho-PKCζ, and Sp1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-PKC, phospho-mTOR^ser2481 (mTORC2), phospho-mTOR^ser2448 (mTORC1), and mTOR were purchased from Cell Signaling (Beverly, MA, USA). The Akt inhibitor I was aquired from Calbiochem (La Jolla, CA, USA). The CD34, GPR40, phospho-Sp1, and MT3-MMP antibodies were obtained from Abcam (Cambridge, MA, USA). Mithramycin A was purchased from Tocris (Bristol, UK). Zymogram gels were bought from Novex (San Diego, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA, USA). All other reagents were used as received and were of the highest purity commercially available.