Figure 3.

Effects of RF-Id on 5-LOX activity in intact cells and homogenates and on 5-LOX translocation. (A) Intact neutrophils were pre-incubated with RF-Id for 15 min, 37°C and stimulated with 2.5 μM A23187 plus 20 μM AA for 10 min at 37°C. Neutrophil homogenates were incubated with RF-Id for 15 min at 4°C. After addition of 1 mM ATP, samples were warmed up for 30 s at 37°C and stimulated with 2 mM CaCl₂ and 20 μM AA for 10 min at 37°C. Data are expressed as percentage of vehicle control (0.1% DMSO); means ± SEM; n = 3. (B) Effects of RF-Id and hyperforin on 5-LOX subcellular localization in neutrophils following stimulation with 2.5 μM A23187 for 10 min at 37°C. The distribution of 5-LOX was analysed by Western blot in the nuclear and non-nuclear fraction of mild detergent (0.1% NP-40)-lysed cells. One representative experiment of three independent experiments is shown. Values are given as arbitrary densitometric units. Data are expressed as percentage of control (the 5-LOX band from unstimulated neutrophils was set to 100% in Non-Nuc, the 5-LOX band from A23187-activated neutrophils was set 100% in Nuc), means + SEM., n = 3.