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. 2015 Feb 24;43(5):2888–2901. doi: 10.1093/nar/gkv110

Figure 6.

Figure 6.

Role of RPS5 in HCV translation and replication. (A) Effect of partial knockdown of RPS5 on HCV IRES activity. Huh7 cells were transfected with siNSP RNA or siRPS5 RNA. 48 h later, cells were again transfected with HCV bicistronic RNA. Cells were harvested 10 h post-transfection (5 h after changing serum free media with DMEM plus serum). Graph represents the average of three independent experiments. (B) Western blot depicting the partial knockdown of RPS5. (C) Inhibition of HCV IRES activity using anti-RPS5 antibody. HCV bicistronic RNA was translated using RRL in the presence of increasing concentration of anti-RPS5 antibody. Non-specific IgG was used as control. (D) Schematic of HCV monocistronic replicon RNA. (E) Western blot depicting the effect of partial knockdown of RPS5 on HCV protein synthesis. Partial knockdown of RPS5 was achieved by transfection of 50 or 100 nM siRPS5 RNA into Huh7 cells harboring HCV sub genomic replicon (Rep2a). siNSP RNA transfection was used as control. Cells were harvested 72 h post-transfection. (F and G) Effect of partial knockdown of RPS5 on polysome profile. Huh7 cells were transfected with 100 nM siNSP or siRPS5 RNA. After 96 h, cells were harvested in lysis buffer and lysate was fractionated on a 15–50% sucrose gradient. Polysome profile was obtained by plotting O.D. 260 of each fraction against the distance (mm). (H) Western blot indicating the partial knockdown of RPS5 protein. (I) Effect of partial knockdown of RPS5 on abundance of 40S and 60S subunits in the cell. Huh7 cells were transfected with 100 nM siNSP or siRPS5 RNA. After 96 h cells were harvested in lysis buffer and lysate was fractionated on a 5–30% sucrose gradient containing 25 mM EDTA. Profile was obtained by plotting O.D. 260 against distance (mm). This is a representative profile of three independent experiments. (J) Western blot indicating the partial knockdown of RPS5 protein. (K) Effect of partial knockdown of RPS5 on protein synthesis by L-[35S]-methionine incorporation. Cells were transfected with 50 and 100 nM of either siNSP or siRPS5 RNA. After 48 h, 35S-methionine incorporation assay was performed and the percentage incorporation was plot. Graph represents the average of three independent experiments. (L) Western blot depicting the partial knockdown of RPS5.