![Figure 1. Nuclear KDR expression in primary acute myeloid leukemias and cell lines. (A) Western blot analysis of cytosolic (C) and nuclear (N) protein extracts from HEL, immunoprecipated with phosphotyrosine Abs, and stained against KDR and phosphotyrosine. Note the intense KDR expression, in its phosphorylated form, in nuclear protein extracts of untreated HEL cells. These results are representative of 3 independent experiments. (B) Immunofluorescence analysis of HEL, HL-60 and 3 primary leukemias in serum free medium. These cells were stained for KDR (PE) and DNA (nucleus) (DAPI [4′,6-diamidino-2-phenylindole]). Note the nuclear expression of KDR in all samples shown. This result is seen in most (95%) cells in each experiment, and was obtained at least 3 times. Negative controls are included (primary Ab alone and secondary Ab alone). Original magnification, × 630. (C) Confocal microscopy analysis, demonstrating KDR is predominantly located to the nucleus of HEL cells: intermediate sections, corresponding to the nucleus, have intense staining compared with the most superficial (cytosolic) sections. Confocal microscopy was performed as described in “Materials and methods.” Results were obtained at least 3 times in independent experiments. (D) Immunofluorescence analysis of HEL, HL-60, and 2 primary leukemias cultured in the presence of the external blocker Ab 4.6.1 or the internal KDR inhibitor for 12 hours. Cells treated with the external blocker demonstrated a clear shift in KDR (PE) localization, now seen at the cell surface. In contrast, the internal inhibitor has a minor effect in KDR localization (clear nuclear staining is still seen) in all samples. Nuclear staining is shown in blue (DAPI). As above, this result is seen in most (90%) cells in each condition. Original magnification, × 630. (E) Immunofluorescence analysis of HEL cells in culture, stained for FLT-1 (PE) and nuclear staining (DAPI). Note the expression of FLT-1, seen only at the cell surface (cytosol and membrane), even when cells are treated with the external VEGF blocker Ab 4.6.1. Results are representative of at least 95% of cells in culture, and were obtained in 3 independent experiments. Original magnification, × 630.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/10/10.1182_blood-2003-05-1634/6/zh80100461180001.jpeg?Expires=1735550284&Signature=ewrhtPU561BtyMF6YejfUHhp1oO9362qNpjNg46p6BUCkoLHSl~9UwNYYdt5hs5KiqiBZfmt7fWmTElUxL15w~~vNly5u7kJN9WNhf7IW7LCaB2oWeMZYNDAdP1wOm~Qkm7xkWfl37EFBK7IzTBVZEWiHxx7AF~8vXZf2td1ahiiMWQ-AWN3KKkk1qJRieiSr9rES7N3gOEtCinQ8wrfXZC0Mjuvgmfv9JW73NRFB7d2Y6NRo83zp~WmMYuxylefTI8VTOP3Hsec3t08-NmEqBazJIwvO3~7H1LDHrAl~XWIAxX67-17x1dFQdZzyIuV8vIN2XxHpZih2Q2XgcgziA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Nuclear KDR expression in primary acute myeloid leukemias and cell lines. (A) Western blot analysis of cytosolic (C) and nuclear (N) protein extracts from HEL, immunoprecipated with phosphotyrosine Abs, and stained against KDR and phosphotyrosine. Note the intense KDR expression, in its phosphorylated form, in nuclear protein extracts of untreated HEL cells. These results are representative of 3 independent experiments. (B) Immunofluorescence analysis of HEL, HL-60 and 3 primary leukemias in serum free medium. These cells were stained for KDR (PE) and DNA (nucleus) (DAPI [4′,6-diamidino-2-phenylindole]). Note the nuclear expression of KDR in all samples shown. This result is seen in most (95%) cells in each experiment, and was obtained at least 3 times. Negative controls are included (primary Ab alone and secondary Ab alone). Original magnification, × 630. (C) Confocal microscopy analysis, demonstrating KDR is predominantly located to the nucleus of HEL cells: intermediate sections, corresponding to the nucleus, have intense staining compared with the most superficial (cytosolic) sections. Confocal microscopy was performed as described in “Materials and methods.” Results were obtained at least 3 times in independent experiments. (D) Immunofluorescence analysis of HEL, HL-60, and 2 primary leukemias cultured in the presence of the external blocker Ab 4.6.1 or the internal KDR inhibitor for 12 hours. Cells treated with the external blocker demonstrated a clear shift in KDR (PE) localization, now seen at the cell surface. In contrast, the internal inhibitor has a minor effect in KDR localization (clear nuclear staining is still seen) in all samples. Nuclear staining is shown in blue (DAPI). As above, this result is seen in most (90%) cells in each condition. Original magnification, × 630. (E) Immunofluorescence analysis of HEL cells in culture, stained for FLT-1 (PE) and nuclear staining (DAPI). Note the expression of FLT-1, seen only at the cell surface (cytosol and membrane), even when cells are treated with the external VEGF blocker Ab 4.6.1. Results are representative of at least 95% of cells in culture, and were obtained in 3 independent experiments. Original magnification, × 630.
