Fig. 1.

IGFBP-3 forms a complex with NONO and SFPQ in response to etoposide treatment. a MDA-MB-468 basal-like TNBC cells were exposed to 20 µM etoposide (Etop) for the indicated times, and IGFBP-3-interacting proteins precipitated from cell lysates by anti-IGFBP-3 antiserum (Fab fraction) coupled to agarose beads. Uncoupled agarose beads were used for IP controls. Samples were blotted for NONO and SFPQ after fractionation by SDS-PAGE. Panels on right show blots of whole-cell lysates without IP. Molecular weight markers are shown on the left. b IGFBP-3 was downregulated in MDA-MB-468 cells by siRNA, and cell lysates IP’d with IGFBP-3-Fab beads 2 h after etoposide stimulation. Precipitates were blotted for NONO, SFPQ, and IGFBP-3. c MDA-MB-468 cells were treated with etoposide, and nuclear extracts were prepared and immunoprecipitated with IGFBP-3-Fab beads. Panels on left show SFPQ, NONO, lamin B1 (nuclear marker), and GAPDH (cytoplasmic marker) in whole nuclear extracts (5% of immunoprecipitated sample). GAPDH in the whole cytoplasmic fraction, run on the same gel, is also shown for comparison. Panels on right show the same proteins after IP. Only 0 and 4 h time points are shown from a 4-h timecourse. For each analyte, all samples (input and IP) were run on the same gel. For SFPQ and NONO, but not Lamin B1 or GAPDH, the input blots shown were from shorter exposures, to avoid saturating the images. d Similar experiment to that shown in Fig. 1a, but in HCC1806 basal-like TNBC cells. e Quantitation of bands immunoblotted for NONO and SFPQ in HCC1806 cells. Data are mean band density ± SEM from 5 experiments. *P < 0.05 vs. time 0 by post hoc Fisher’s LSD test after ANOVA. f Binding of recombinant IGFBP-3 to immobilized recombinant NONO, measured in an ELISA format in which bound IGFBP-3 is immunodetected and quantitated colorimetrically at 450 nm. See Methods for details. Panels show dose–response curves for NONO (left) and IGFBP-3 (right)