Fig. 4.

RIG-I plays a key role in MX1 expression induced by miR-1248 in phSG cells. (a) Western blot analyses of MX1 and ISG15 in phSG cells transfected with the indicated siRNAs together with miR-1248 mimic showed that only knockdown of RIG-I and IFNAR1/R2 abolished MX1 induction (lanes 5 and 6). Knockdown of MAVS attenuates MX1 induction (lane 4). (b) Transduction of pAC-Pre-miR-1248 also induced MX1 expression and this effect is abolished upon knockdown of DICER1 and RIG-I. pAC-EGFP was used as viral transduction control. (c) Western blot analysis of MX1 and RIG-I indicates that transduction of pAC-Pre-miR-1248 induced expression of both proteins in phSG cells (lanes 6), but not in the HSG cell line (lane 2). pAC-EGFP (lane 1, 5) served as a negative control for viral transduction and a RIG-I expression construct (pEF-Flag-RIG-I, lane 3) was used as a positive control. β-actin was used for loading control (A, B). (d) Protein-miRNAs complex was immunoprecipitated by GFP, RIG-I or AGO2 antibody and the level of miR-1248 was quantified by RT-qPCR, showing direct interaction of miR-1248 with both RIG-I and AGO2. Data are shown as fold change (mean ± S.E., average from three independent experiments) over the GFP antibody control. Tx: transfection.