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. Author manuscript; available in PMC: 2015 May 21.
Published in final edited form as: Mol Cell Biochem. 2009 Jul 9;332(0):173–181. doi: 10.1007/s11010-009-0188-0

Figure 3. In vivo gene knockdown of Kap7 prevents accumulation of PRH in the nucleus.

Figure 3

a. Hek 293 cells were co-transfected with a plasmid coding for Xpress-tagged PRH along with either siRNAs targeting Kap7 (panel a, i and ii) or Kapβ1 (panel a, v and vi). Additionally Hek 293 cells were co-transfected with a plasmid coding for GFP-NLS along with either siRNAs targeting Kap7 (panel a, iii and iv) or Kapβ1 (panel a, vii and viii). 96 hours post transfection, cells were washed, fixed, and stained with DAPI (Note that the DAPI stained cells depicted in panels a, i, iii, v, and vii are the same cells as in a, ii, iv, vi, viii, respectively). Immunofluorescence for Xpress-tagged PRH is shown in panels a, ii and vi. Depletion of Kap7 is associated with a striking decrease in the nuclear accumulation of PRH (panel a, ii) as compared to cells transfected with siRNA targeted to deplete Kapβ1 (panel a, vi). Fluorescence for GFP-NLS is shown in panels a, iv and viii. Depletion of Kap7 had no effect on the nuclear accumulation of GFP-NLS (panel a, iv). In contrast, cells transfected with siRNA targeted to deplete Kapβ1 (panel a, viii) exhibited reduced nuclear accumulation of GFP-NLS and a noticeable increase in cytoplasmic accumulation. b. Hep G2 cells were transfected with siRNAs targeted to Kap7 or Kapβ1. 96 hours post transfection, cells were washed, fixed and immunofluorescence for endogenous PRH was performed. Depletion of Kap7 is associated with a striking decrease in the nuclear accumulation of PRH (panel b, ii) as compared to control cells that were not transfected with siRNA (Mock) (panel b, i), or cells transfected with siRNA targeted to deplete Kapβ1 (panel b, iii). c. Quantitative RT-PCR utilizing gene specific DNA primers for Kap7 and Kapβ1 at 72 hours post siRNA transfection. Kap7 mRNA was significantly reduced in cells that were transfected with Kap7 siRNA as compared to a mock transfection, or cells transfected with Kapβ1 siRNA (n = 3, *p<0.005 relative to the mock transfection group). Additionally cells transfected with Kapβ1 siRNA, exhibited a significant reduction of Kapβ1 mRNA as compared to a mock transfection or cells transfected with Kap7 siRNA (n = 3, *p<0.005 relative to the mock transfection group). Error bars represent standard error of the mean (SEM). d. Western blotting using protein specific antibodies confirmed a significant reduction of Kap7 protein in cells that were transfected with Kap7 siRNA, but not cells transfected with Kapβ1 siRNA, or a mock transfection (n = 3, *p<0.005 relative to the mock transfection group). Additionally cells transfected with Kapβ1 siRNA, did not show a reduction of Kap7 protein, but did show a significant reduction of Kapβ1 protein (n = 3, *p<0.005 relative to the mock transfection group). Error bars represent SEM. e. Representative images for western blot data for experiment in c. Rows represent western blot data using respective antibodies against Kapβ1, Kap7 and GAPDH. All samples were normalized against the GAPDH loading control. Columns represent samples treated with siRNAs for Kapβ1 (β1), Kap7 (7), or the mock transfection (M).