TNF-α enhances fMLF-induced Pin1 and p47phox translocation to the membranes in human neutrophils and the effect of juglone. (A) Neutrophils were treated with TNF-α (20 ng/mL), fMLF (10−7M), or TNF-α followed by fMLF (TNF-α + fMLF) and then lysed by nitrogen cavitation. Membranes and cytosols were separated by ultracentrifugation on a sucrose gradient. Proteins were analyzed by SDS-PAGE and Western blotting with anti-Pin1, anti-p47phox, and anti-pSer345 antibodies. (B) Neutrophils were treated with TNF-α, fMLF, or TNF-α + fMLF in the absence or presence of 250nM juglone and then lysed. Membranes were separated by ultracentrifugation on a sucrose gradient, and proteins were analyzed by SDS-PAGE and immunoblotting (IB). (representative of 7 experiments).
