IGFBP-2 DAPI Compositex-axis x-axis x-axis

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Figure S1A

Figure S1B:

(S1A) 3D confocal analysis of IGFBP-2 immunofluorescence in LNCaP and MCF-7 cells. Confirmation that nuclear IGFBP-2 staining is due to intranuclear, and not cell membrane bound, IGFBP-2. (S1B) 3D animations of IGFBP-2 immunofluorescence in LNCaP cells showing intranuclear localization of IGFBP-2.

S2. Import assay of IGFBP-3 was carried out, in parallel to the IGFBP-2 import assay, as a positive control. As previously described, IGFBP-3 requires ATP and cytosolic factors for nuclear translocation. Digitonin-permeabilized SHEP cells were incubated with full length IGFBP-3 in the presence of cytosol and an ATP-regenerating system. We examined depletion of ATP and cytosol from the import assay. Transport studies were also carried out with the addition of anti-importin α/β in the presence or absence of RRL respectively. Images are representative of at least three independent experiments. Cells were visualised by a laser scanning confocal microscope (Leica TCS SP2 SE, objective X40).

Figure S2

Figure S3

(+) IgG

(+) IgG

Anti-IGFBP-3 DAPI Composite

Anti-IGFBP-2 DAPI Composite

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S3 . Transport studies were carried out with the addition of anti-importin α/β in the presence or absence of RRL respectively, to determine their function in the nuclear import of IGFBP-2 and -3. IgG was used as a negative control to demonstrate the specific blocking effects of anti-importin α and β. Images are representative of at least three independent experiments. Cells were visualised by a laser scanning confocal microscope (Leica TCS SP2 SE, objective X40).

WIB IGFBP-232 kDa

WIB Importin

60 kDa

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IP IGFBP-2 IP Importin-

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IP IGFBP-2

32 kDa

Figure S4

Figure S4. IGFBP-2 /Importin complexes: The presence of IGFBP-2/importin-α complexes in SHEP cells over-expressing IGFBP-2 was demonstrated by immunoprecipitation as described in Materials and Methods. The IP with anti-IGFBP-2 resolved immunoreactive bands (Bound) for IGFBP-2 (Top panel; 32kDa band) and importin-α (Bottom panel; 60kDa band). Cell extract starting material (Input) and post IP material (Unbound) and purified IGFBP-2 (10 ng) were also include as controls. The right panel shows, in reduced scale, lanes of the WIB for the Input and Unbound samples to provide evidence for specificity of the anti-IGFBP-2 antibody (32 kDa band). Correspondent lanes for Input and Unbound samples in the top and in the right panels (full size immunoblotting membrane) are demarked by a dotted red line.