RT-qPCR analysis of socs3 expression

Transfected HEK293T cells were incubated in SF-DMEM (3 h) before treatment without or with OSM (10 ng/ml, 45 min) and RNA extraction (ReliaPrep RNA Cell Miniprep System, Promega). cDNA was generated using oligo(dT)₂₀ primer (GoScript Reverse Transcription System, Promega), before qPCR using primers for socs3 and gapdh, and iTaq Universal SYBR Green Supermix (Bio-Rad). Socs3 expression was normalised to gapdh using the comparative C_T method [59,62], and then calculated relative to that for control (GFP or GFP-RABV N)-expressing cells treated with OSM. Data from 4 independent assays were combined, where the value from each assay is the mean of technical replicates. Primer sequences were: 5’-GGAGTTCCTGGACCAGTACG-3’ and 5’-TTCTTGTGCTTGTGCCATGT-3’ for socs3; 5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GGTCATGAGTCCTTCCACGAT-3’ for gapdh.