Figure 4.

MEX3A expression affects RIG-I protein stability. (A,B) Representative immunoblotting (A) and densitometric analysis (B) of GFP-RIG-I protein level in HEK293T cells transfected with increasing amounts of Myc-tagged MEX3A, MEX3C and RNF122, in a (1:1) and (1:0.5) ratio, compared to the amount of the transfected GFP-RIG-I. (C) HEK293T cells were transfected with equal quantities of GFP-RIG-I and Myc-tagged MEX3A and MEX3C, alone or in combination. The levels of the indicated protein were assessed by immunoblot analysis. (D) Densitometric analysis of GFP-RIG-I protein levels shown in (C) and normalized on actin levels. (E) HEK293T cells were co-transfected with GFP-RIG-I, an empty vector as control or Myc-MEX3A, in a ratio (1:0.5). Twelve hours after transfection, GFP-RIG-I half-life was assayed, following treatment with CHX (100 µg/mL), at the indicated time. (F) Densitometric analysis of RIG-I protein levels shown in (E) and normalized on actin levels. (G) Protein expression levels of endogenous MEX3A and RIG-I in A-172 and T98G GB cell lines transduced with lentiviral vectors encoding either control shRNA (shCTR) or MEX3A shRNAs (shMEX3A#1 and shMEX3A#2). (H) Densitometric analysis of RIG-I and MEX3A protein levels shown in (G) and normalized on actin levels. (I) Half-life of endogenous RIG-I in T98G cells infected with lentiviral particles encoding shCTR or shMEX3A#2. Cells were treated with CHX (50 µg/mL) at the indicated time points. (J) Densitometric analysis of the RIG-I protein levels shown in (I). All the densitometric analysis represent the mean of protein levels of three independent experiments. Mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001 calculated with two-sided Student’s t-test.