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. 2016 Feb 18;6:21410. doi: 10.1038/srep21410

Figure 5.

Figure 5

Extracellular importin α1 affects cell proliferation via interaction with FGF1 (a) GST, GST-FGF1, GST-FGF2, or GST-IGFBP5 (50 pmol) were incubated with 3 × Flag-importin α1 (50 pmol) immobilized on glutathione beads, and subjected to immunoblot analysis with the indicated antibodies. (b) Starved HCT116 cells were incubated in PBS or FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 at the indicated concentrations for 48 h. Cell proliferation was measured using Cell Counting Reagent. Data are means ± SD from three independent experiments. *P < 0.005. (c) The starved cells were incubated in PBS or FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 for 10 min, and subjected to immunoblot analysis with indicated antibodies. (d) Starved HCT116 cells were incubated in FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 (20 ng/ml) for the indicated time courses, and subjected to immunoblot analysis with indicated antibodies. (e) HCT116 cells were transfected with control or importin α1 siRNAs, followed by starvation and incubation in PBS or FGF1 or FGF2 (20 ng/ml) in the presence or absence of recombinant importin α1 (20 ng/ml) for 10 min, and subjected to immunoblot analysis with indicated antibodies. (f) GST-FGF1 (50 pmol) was incubated with 50 pmol 3 × Flag-importin α1 immobilized on glutathione beads in the presence of anti-importin α1 monoclonal antibody (mAb) or control mouse IgGs (as control), and subjected to immunoblot analysis. (g) Starved HCT116 cells were incubated in FGF1 (20 ng/ml) in the presence or absence of recombinant importin α1 (20 ng/ml) with anti-importin α1 mAb (25 or 250 ng/ml) or control mouse IgGs for 10 min, and subjected to immunoblot analysis with indicated antibodies. (h,i) Cell proliferation of HCT116 cells (h) and AGS cells (i) were measured for the indicated time courses after treatment without or with normal mouse IgGs as isotype control or anti-importin α1 mAb (250 ng/ml), in triplicate for each condition, using Cell Counting Reagent. Data are means ± SD from three independent experiments. *P < 0.005.