Figure 1. PCNA is expressed exclusively... | Rockefeller University Press

$Figure 1. PCNA is expressed exclusively in the cytosol of mature neutrophils. (A) PCNA immunodetection in neutrophils (PMN), lymphocytes (Ly), or PLB985 promyelocytic cells. 50,000 cells/lane were analyzed using PC10 mAb as the primary antibody; anti–β-actin served as the loading control. (B) PCNA expression in different neutrophil subcellular compartments with β-actin, human neutrophil elastase, or lamin B serving as control markers for the cytosolic (Cyt), granular (Gr), and nuclear (Nu) fractions, respectively. The SDS–PAGE gel was run using 50 µg protein/lane, and PCNA was detected with the PC10 mAb. (C) Immunofluorescence analysis by confocal microscopy of PCNA localization in human CD34+ cells before (CD34+) and at different times (7 or 13 d) during granulocyte differentiation and in mature neutrophils using the rabbit pAb Ab5 and TO-PRO 3 iodide for nuclear labeling. Bar graphs show percentages of cells exhibiting nuclear, mixed nuclear–cytoplasmic, or cytoplasmic PCNA localization, as determined by counting the cells under the microscope. (D) Immunofluorescence analysis by confocal microscopy of PCNA localization in human BM cells after sorting on a Percoll gradient. Cells from band 3, containing myeloblasts and promyelocytes (PM; top), were labeled with MPO. In contrast, cells from band 1, containing the mature neutrophils as shown by their typical nuclear morphology (PMN; bottom), were labeled with anti-CD35. A–D show representative experiments of three yielding the same results. Bars, 10 µm.$

Figure 1.

PCNA is expressed exclusively in the cytosol of mature neutrophils. (A) PCNA immunodetection in neutrophils (PMN), lymphocytes (Ly), or PLB985 promyelocytic cells. 50,000 cells/lane were analyzed using PC10 mAb as the primary antibody; anti–β-actin served as the loading control. (B) PCNA expression in different neutrophil subcellular compartments with β-actin, human neutrophil elastase, or lamin B serving as control markers for the cytosolic (Cyt), granular (Gr), and nuclear (Nu) fractions, respectively. The SDS–PAGE gel was run using 50 µg protein/lane, and PCNA was detected with the PC10 mAb. (C) Immunofluorescence analysis by confocal microscopy of PCNA localization in human CD34⁺ cells before (CD34⁺) and at different times (7 or 13 d) during granulocyte differentiation and in mature neutrophils using the rabbit pAb Ab5 and TO-PRO 3 iodide for nuclear labeling. Bar graphs show percentages of cells exhibiting nuclear, mixed nuclear–cytoplasmic, or cytoplasmic PCNA localization, as determined by counting the cells under the microscope. (D) Immunofluorescence analysis by confocal microscopy of PCNA localization in human BM cells after sorting on a Percoll gradient. Cells from band 3, containing myeloblasts and promyelocytes (PM; top), were labeled with MPO. In contrast, cells from band 1, containing the mature neutrophils as shown by their typical nuclear morphology (PMN; bottom), were labeled with anti-CD35. A–D show representative experiments of three yielding the same results. Bars, 10 µm.

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