Figure 3

Depletion of PI(4,5)P ₂ and it’s effect on BTV production. (A) Western Blot analysis of HeLa (right) and BSR (left) cells transfected with myc-tagged 5ptaseIV (lane 1) or Δ1 mutant (lane 2) and infected with BTV-1 for the expression of viral (VP5 and NS2) and cellular (actin and tubulin) proteins 4 and 12 hrs post-infection. Controls consist of HeLa cells that have not been transfected but infected with BTV-1 (3) and cells that have neither been transfected nor infected (4). The proteins were detected by specific antibodies. The western blots were repeated 2 times on three independent experiments. (B and C) Analysis of relative (B) and total (C) virus titres in infected HeLa (right) and BSR (left) cells in the presence of 5ptaseIV (IV), Δ mutant (Δ), transfection reagent only (T) or absence of any plasmid (−). Cells infected with BTV-1, 12 hrs post-transfection with either 5ptaseIV or Δ mutant were harvested at 4 and 12 hrs post-infection. For the relative virus tires, the total titre for each time post-infection was normalized to 100% for untreated cells. Bars indicate the standard error of three replicates of the experiments.