IF proteins lamin B1 and keratins colocalize with endogenous and exogenous APC in cultured cells. (A) Confocal immunofluorescence microscopy reveals partial overlap of APC (red) and keratin (green) or lamin B1 (green) in HCT116βw cells. Single projections of 20 images resolved along the z-axes for lamin and 30 images for keratin are shown. Scale bar, 4.6 μm. (B) More than 700 individual images were analyzed for each protein pair examined to determine the Pearson's correlation coefficient (upper graph) or the Manders overlap coefficient (lower graph). Additional file 4B shows representative image of the negative control (both secondary antibodies with no primary antibody). The average Pearson's correlation coefficient ± SEM calculated from n individual z-series images (n = 900, keratin/APC; n = 720, lamin B1/APC) were significantly higher than the correlation coefficient determined for the negative control (* and ** p < 0.0001). (C) The Van Steeples cross correlation function (CCF) was plotted as a function of image offset in pixels. Keratin and lamin B1 each show a peak of correlation with the APC signal in the original position with no offset (dx, red line). In contrast, the CCF is low, with multiple peaks for keratin or lamin B1 with a randomized APC signal. (D) HCT116βw cells expressing full-length APC fused to GFP were stained for keratin or lamin B1 (red). Confocal images show partial overlap of the keratin and lamin B1 signals with GFP. Scale bars, 5 μm.