Activation of Human Neutrophils by the Anti-Inflammatory Mediator Esenbeckia leiocarpa Leads to Atypical Apoptosis
Figure 2
CHE induces tyrosine phosphorylation events in human neutrophils. (a), Freshly PMNs were isolated and incubated (10 × 106 cells/mL) with the diluent (HBSS-1% DMSO, Ctrl), granulocyte macrophage colony-stimulating factor (GM) (65 ng/mL) (lane 2), and crude hydroalcoholic extract (CHE) (500 μg/mL) at the periods of stimulation of 0.5, 1, 5, 15, 30, 45, or 60 min. Immunoblotting was performed as described in Section 2. Top panel: tyrosine phosphorylation of intracellular proteins; bottom panel, Coomassie blue staining of the membrane to indicate equivalence in loading. (b) and (c) cells were stimulated for 10 min with the diluent (Ctrl), GM-CSF (GM, 65 ng/mL), or with CHE (500 μg/mL) for 5, 15 or 30 min. Immunoblotting was performed as described in Section 2. (b) and (c) Top panels illustrate the membranes that were revealed with antiphosphorylated p38 (b) or antiphosphorylated ERK-1/2 (c) antibodies; bottom panels are the corresponding membrane stained with the unphosphorylated form of p38 or ERK-1/2, respectively, indicating equivalent loading. Results shown are representative of at least three different experiments.
