Sybr green detection system

RPMI-1640 medium, fetal bovine serum (FBS) and penicillin were purchased from Invitrogen; Thermo Fisher Scientific. Antibodies against TPD52, transforming acidic coiled-coil-containing protein 3 (TACC3), Ephrin A1(EFNA1), transferrin (TRF) and β-actin were obtained from Abcam. DNAJB1, LDH, trypsin and goat anti-rabbit/mouse secondary antibodies conjugated to horseradish peroxidase were from Sigma-Aldrich. The Total RNA extraction kit, SYBR-Green detection system and the Bradford protein assay kit were purchased from Tiangen Biotech. The ultrafiltration device, ECL reagent and polyvinylidene difluoride (PVDF) were from Millipore. The DAB detection kit was from Maixin Biotech. The iTRAQ labeling Reagents kit was from Applied Biosystems.

Total RNA was extracted and reverse transcribed from the cultured cells using RNA simple total RNA extraction kit (Tiangen Biotech) according to the instructions of the manufacturer as previously described (13 (link)). qPCR was performed using the SYBR-Green detection system (Tiangen Biotech). The primers for qPCR reactions are listed in Table SI. The relative expression levels of the target genes were according to GAPDH. The qPCR thermocycling conditions were as follows: 95°C for 30 sec, 45 cycles of 90°C for 30 sec, 60°C for 20 sec, 72°C for 25 sec, and 72°C for 15 min. The relative gene expression levels were analyzed using the 2^−∆∆Cq method (14 (link)). All experiments were performed in triplicate.