Fig 1: Subcellular localization of DOK1 protein variants. (A) Anti-N Ab (a-N) recognizes nuclear DOK1, anti-C Ab (a-C) cytoplasmic DOK1. Cycling cells were fixed and stained for immunofluorescence microscopy. Data show enrichment of DOK1 signals detected by a-N Ab in the nucleus (NUC) as -fold ± S.E. compared to a-C Ab (n = 3 per cell line, > 30 nuclei per field, n = 5 fields, *p < 0.05 a-N vs. a-C; Mann Whitney test). Colors: green = DOK1, blue = nuclei; Magnification 630 ×. (B) Anti-N Ab detects p37 DOK1 in the nucleus of malignant cells. Cells were subjected to subcellular fractionation and Western blotting. O.D. values from bands in gels are means ± S.E. of DOK1 protein per fraction (n = 3 per cell line, *p < 0.05 vs. NUC; Kruskal Wallis test). Legend: CYT = cytoplasm, NUC = nucleus, INS = insoluble membrane and matrix fraction (cytoskeleton, chromatin e.a.). (C) Anti-C Ab detects colocalization of cytoplasmic DOK1 with calnexin (ER marker) but not with POM121 (ENV marker) in the perinuclear area of the endoplasmic reticulum (ER) and the nuclear envelope (ENV). Anti-N Ab detects overlay of nuclear DOK1 with nuclei (DAPI). HEK293T cells were analysed as in A. Colors: green = organelle marker, red = DOK1, blue = nuclei; Magnification 630 ×. (D) Anti-C Ab detects FL p62 and p44 DOK1 in the cytoplasm and insoluble fraction of non-malignant cells. Fresh-frozen normal colon (NC) tissue from patients or C57BL6J mice was subjected to fractionation as in B. Results are shown as in B (n = 3 per species, *p < 0.05 vs. NUC, Kruskal Wallis test).