LTB₄ elicits translocation and phosphorylation of p47phox in a PKC-δ-dependent manner. (A) Rat AMs (4 × 10⁶/well) were stimulated with 1 nM LTB₄, 100 nM LTC₄, 100 nM LTD₄, and 1000 nM 5(S)-HETE or 10 nM PMA for 5 minutes and then were harvested and fractionated as described in “Materials and methods.” Immunoblotting of membrane fractions was performed using anti-p47phox (1:500). Results are representative of those from 3 experiments. (B) Rat AMs (4 × 10⁶/well) were pretreated with 10 nM RO32-0432 (PKC-α inhibitor), 6 μM rottlerin (PKC-δ inhibitor), or vehicle control for 20 minutes, stimulated with 1 nM LTB₄ for 5 minutes, and then harvested and fractionated as described in “Materials and methods.” The cytosolic fraction was immunoprecipitated, and immunoprecipitated protein was immunoblotted using antiphosphoserine (1:900). The total cytosolic fraction and the membrane fraction were probed for total p47phox (1:500). Results are representative of those from 2 experiments. Densitometry analysis is from phosphorylated p47phox or membrane-translocated p47phox. In all instances, density values of bands were corrected by subtraction of the background values. Numbers under lanes indicate relative density of phosphorylated or membrane-translocated p47phox. To ensure equal protein loading, blots were stripped and reprobed for FLAP.