Figure 1

Generation of stable imp7 knock down (KD) cells. (A) Left panels: Western blot with an anti-imp7 antibody and an anti-Ran antibody on total cell extracts obtained from a low passage polyclonal population of DxR KD (lane 1) and imp7 KD HeLa cells (lane 2), a polyclonal population of DxR KD (lane 3), imp7KD (lane 4) and the same imp7 KD population expressing an imp7 cDNA with two silent mutations making it resistant to the shRNA (imp7+7R) (lane 5). Note that cells in lanes 3 to 5 were grown for 4 weeks to allow for selection of the imp7+7R line. Right panel: Western blot with an anti-imp7 antibody and an anti-Ran antibody on total cell extracts obtained from a polyclonal population of DxR KD cells (lane 1) and imp7 KD Jurkat cells (lane 2). (B) DxR KD (shDxR), imp7 KD (shimp7) and imp7 back complemented (shimp7+7R) HeLa cells were plated onto 24-well plates to have the same confluency by the next day and infected with five fold serial dilutions of a VSV-G pseudotyped HIV-1 vector (pHR') expressing GFP. Twenty-four hours after infection the percentage of GFP+ cells was measured by flow cytometry. (C) Cells were infected with three fold serial dilutions of a VSV-G pseudotyped SIVmac vector expressing GFP and analysed as in (B). Similar results were obtained in least three independent experiments using different virus stocks. (D) Western blot with an anti-imp7 antibody and an anti-Ran antibody on total cell extracts obtained from a polyclonal population of DxR KD HeLa cells (lane 1) and three different imp7 KD clones (lane 2, clone 2; lane 3, clone 4; lane 4, clone 8). The bands were scanned and the intensity of the imp7 band relative to Ran is shown below each sample. (E) DxR KD cells and imp7 KD clonal cell populations were infected with a VSV-G pseudotyped HIV-1 vector (pHR') or SIVmac vector expressing GFP at an MOI of 0.03 and the percentage of GFP+ cells counted 24 hours after infection by flow cytometry. Data are expressed as average percentage of infection relative the parent line control (shDxR) ± SD of three independent experiments. (F) DxR KD and imp7 KD Jurkat cells were infected with three fold serial dilutions of a VSV-G pseudotyped HIV-1 vector (pCSGW) expressing GFP and the percentage of GFP+ cells counted 24 hours after infection by flow cytometry. Similar results were obtained in two additional experiments using different virus stocks.