Turbofect

For the promoter activity assay, the cells were co-transfected with the proper amount of SOCS5: rs3814039_G/pGL4.17 or SOCS5: rs3814039_C/pGL4.17 together with pRL-SV40 Renilla luciferase reporter (Promega, for internal control) using TurboFect™ (Origene) at the basal level or induced by EGF or IL-6. At 48 hours post-transfection, both firefly and Renilla luciferase activity were determined by the Dual luciferase assay system (Promega) following the manufacturer's instructions. For the assay of miRNA target expression, the ESCC cells were transiently transfected with SOCS5: rs3768720_C/pmirGLO or SOCS5: rs3768720_A/pmirGLO using TurboFect™. Firefly and Renilla luciferase activity were both analyzed as described above.

To transiently over express GFP-SOCS5 and myc-tagged EGFR (myc-EGFR) in HEK293T or ESCC cells, the cells were seeded overnight and transfected with indicated amounts of GFP-SOCS5/ pCMV6-AN-GFP and/or pcDNA6A-EGFR plasmids, a kind gift from prof. Mien-Chie Hung 35 (link), using TurboFect™ (Origene) according to manufacturer's instructions. At the indicated time post-transfection, the cells were harvested for protein extraction and functional assay. For the proteasome inhibitor treatment, the cells were treated with 0 (solvent control), 1, and 10 µM of MG132 (Sigma) at 24 hours post-transfection and incubated for 24 hours.