Figure 4. Effect of Rab1b or ARF5 depletion on IRES activity.

(A) The levels of Rab1b and ARF5 were determined by Western blot using anti-Rab1b or anti-ARF5 in comparison with siRNAcontrol-transfected cells. Tubulin is used as loading control. Rab1b- and ARF5-depleted cells were used to monitor relative IRES-dependent translation using bicistronic constructs. The effect on protein synthesis was calculated as luciferase activity/chloramphenicol acetyl transferase activity relative to the control siRNA. Each experiment was repeated three times. Values represent the mean ± SD. Asterisks (P = 0.0024) denotes statistically significant differences between cells treated with the siRNAcontrol and siARF5 RNA. (B) GFP-Rab1b and GFP-ARF5 colocalize with the Golgi compartment, but expression of the dominant-negative GFP-Rab1b DN disorganizes the Golgi. Representative images of HeLa cells transfected side by side with GFP-Rab1b, GFP-Rab1b DN, or GFP-ARF5; fixed 30 h post-transfection; and permeabilized. Immunostaining of the Golgi was carried out using anti-GM130 antibody (bar = 10 μm). White arrows denote colocalization of GM130 and GFP-tagged proteins Rab1b or ARF5 in transfected cells, whereas white asterisks denote GM130 signals in nontransfected cells. Manders’ coefficient obtained for the quantitation of colocalization of GM130 with GFP-tagged proteins (M1) or the reverse (M2) is shown in the bottom panel. (C) Expression of the dominant-negative Rab1b DN affects IRES-dependent translation. Luciferase activity (RLU/μg of protein) measured in triplicate assays using HeLa cells transfected with Rab1b or Rab1b DN and pIRES-luc (P = 0.035) or pCAP-luc (P = 0.190).