Fig. 5.

CLP and FLAP are required for association of 5LO with the nucleus during MM6 cell stimulation. Differentiated MM6 cells (control transfected with nontarget shRNA and knockdown cells) were primed with PMA (100 nM) at 37 °C for 10 min, followed by stimulation with ionophore A23187 (5 µM) for another 5 min. After cooling on ice, subcellular fractionation was performed by mild detergent (0.1% Nonidet P-40) lysis (Materials and Methods). Equal protein amounts (40 µg) of nonnuclear and nuclear fractions were analyzed by 5LO Western blot analysis. β-actin and histone H4 served as loading controls of nonnuclear and nuclear fractions, respectively. Similar results were obtained in two additional experiments.