PMA up-regulates 5-LO kinase activation in PMNLs: inhibition by calphostin C.
PMNLs (5 × 107 in 1 mL PGC buffer) were preincubated at 37°C in the presence or absence of calphostin C, as indicated, for 30 minutes under ordinary fluorescent light. (A) Cells were primed with PMA (100 nM) for 10 minutes before stimulation with 0.1 μM A23187 for 3 minutes at 37°C. Incubations were terminated by the addition of the same volume of SDS-b, vortexed and heated at 95°C for 6 minutes. Aliquots corresponding to 0.5 × 106 PMNLs were electrophoresed on a 10% SDS-polyacrylamide gel polymerized with 0.15 mg/mL purified recombinant 5-LO. In-gel kinase assay was performed as described in “Materials and methods.” Positions of standard proteins are indicated. (B) Nonprimed cells were stimulated with 2.5 μM A23187 for 3 minutes at 37°C and analyzed for 5-LO phosphorylation by in-gel kinase assay. Results are representative of 3 separate experiments.
