Anti resistin

Cells were lysed in lysis buffer supplemented with protease and phosphatase inhibitor (MCE). Proteins were quantified by the Bradford assay (HyClone-Pierce). The proteins were then separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were probed with Abs overnight at 4 °C, and then incubated with a horseradish peroxidase-coupled secondary Ab. Detection was performed using a LumiGLO chemiluminescent substrate system. Anti-p-IGF-I receptor β (IRβ) Cell Signal Technology, USA anti-p-protein kinase B (AKT; 1:1000, Proteintech, China), anti-CAP1 (Abcam, USA), anti-resistin (1:1000, Abcam, USA), anti-p-vasodilator stimulated phosphoprotein (VASP; 1:1000; Cell Signal Technology, USA), anti-p-p65 (1:1000; Cell Signal Technology, USA), anti-Na⁺/K⁺-ATPase (1:1000; Cell Signal Technology, USA), anti-β-actin (1:2000, Abmart, USA), anti-GAPDH (1:2000, Abmart, USA) were used.

RPMI-1640 and trypsin were obtained from Hyclone (Florida, USA), puromycin from Sigma (San Francisco, USA), and fetal bovine serum (FBS) and Opti-MEM from Gibco (New York, USA). Trizol and the reverse transcription kit were from Thermo (Massachusetts, USA), SYBR Green Real-time PCR kit from Guangzhou Ruibo Biotech, and Lipofectamine 3000 from Invitrogen (New York, USA). The miR-625 mimic/inhibitor and respective negative controls (NC) and si-resistin sequences, as well as the wild-type (WT) and mutant (MUT) resistin luciferase reporter plasmids were provided by Shanghai Gemma (Shanghai, China). The lentiviruses harboring these sequences were generated by Shanghai Jikai Gene Bios Inc. All primers were synthesized by Shanghai Sangon (Shanghai, China). Mouse anti-Resistin, anti-vimentin and anti-E-cadherin, and rat anti-p-AKT, anti-p-PI3K, anti-Snail, anti-Twist1 and anti-β-actin antibodies were purchased from Abcam (London, UK). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse and anti-rat IgG secondary antibodies were obtained from Guangzhou Jingcai, fluorescent enzyme detection kit from Promega (Beijing, China), and the IHC EnVison two-step kit and diazobenzidine (DAB) colorimetric kit from Beijing Zhongshan (Beijing, China). Other reagents were of analytical grade.