American Society of Hematology

Figure 5.

$Figure 5. Translocation and colocalization of eNOS and 5-LO in HMC-1. (A) Western blot analysis of 5-LO, eNOS, phospho-eNOS (Ser1177), and α-tubulin in subcellular fractions from HMC-1 cells. Cells were stimulated with A23187 (0.1 μM) for the indicated times. Cell fractionation and Western blotting were performed as described in “Materials and methods.” Equal quantities (15 μg) of protein were added to each lane, and the relative band intensities (numbers below the bands) were determined by densitometry (arbitrary units). Results are representative of 3 independent experiments. (B) Colocalization of eNOS and 5-LO in human mast cells. HMC-1 or LAD 2 cells were untreated or stimulated with A23187 (0.1 μM) or IgE cross-linking (1 μg/mL) for 15 minutes, respectively. Cells were fixed in 4% paraformaldehyde and incubated with rabbit anti-5-LO (red) and mouse anti-eNOS (green). Antibody labeling was detected with rhodamine red-conjugated goat antirabbit and BODIPY-conjugated rabbit antimouse antibodies; overlapping fluorescence is indicated in yellow. White arrowheads indicate nuclear localization, white arrows indicate cytoplasmic staining, and white stars indicate nuclear envelope localization. Original magnification × 800; bar = 10 μm.$

Translocation and colocalization of eNOS and 5-LO in HMC-1. (A) Western blot analysis of 5-LO, eNOS, phospho-eNOS (Ser1177), and α-tubulin in subcellular fractions from HMC-1 cells. Cells were stimulated with A23187 (0.1 μM) for the indicated times. Cell fractionation and Western blotting were performed as described in “Materials and methods.” Equal quantities (15 μg) of protein were added to each lane, and the relative band intensities (numbers below the bands) were determined by densitometry (arbitrary units). Results are representative of 3 independent experiments. (B) Colocalization of eNOS and 5-LO in human mast cells. HMC-1 or LAD 2 cells were untreated or stimulated with A23187 (0.1 μM) or IgE cross-linking (1 μg/mL) for 15 minutes, respectively. Cells were fixed in 4% paraformaldehyde and incubated with rabbit anti-5-LO (red) and mouse anti-eNOS (green). Antibody labeling was detected with rhodamine red-conjugated goat antirabbit and BODIPY-conjugated rabbit antimouse antibodies; overlapping fluorescence is indicated in yellow. White arrowheads indicate nuclear localization, white arrows indicate cytoplasmic staining, and white stars indicate nuclear envelope localization. Original magnification × 800; bar = 10 μm.

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