Translocation of 5-LO and up-regulation of 5-LO product formation in MM6 cells.
(A) Western blot analysis of 5-LO in subcellular fractions from MM6 cells. Cells (1 × 107 in 1 mL PGC buffer) were first incubated (primed) for 10 minutes at 37°C with or without PMA (100 nM). Then A23187 (5 μM) or A23187 and AA (5 and 40 μM, respectively) were added as indicated, and the incubations were continued for another 5 minutes. Cell fractionation and immunoblotting were performed as described in “Materials and methods.” Pair-wise samples (nonnuclear, nuclear) correspond to the identical cell numbers. The relative intensities of blot bands were determined by densitometry (arbitrary units). Similar results were obtained in 2 additional experiments. (B) 5-LO product formation in intact MM6 cells (3 × 106 in 1 mL PGC buffer) was determined by HPLC as described in “Materials and methods.” Cells were primed for 10 minutes with PMA (100 nM), mezerein (100 nM), and sodium orthovanadate (1 mM) before stimulation for 10 minutes with A23187 (5 μM) or A23187and AA (5 and 40 μM, respectively), as indicated. Results are given as mean + SE of 3 independent experiments. Studentt test; **P < .01; *P < .05.
