BMCMCBone Marrow Cultured Mast Cells
BMCMCBone Marrow Cultured Murine Mast Cells (immunology)
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Analysis of ERK1/2 Phosphorylation in BMCMCs. For detection of phosphorylated ERK1/2, C57BL/6-derived BMCMCs were stimulated with 50 ng/mL PMA plus 10 [micro]M A23187 calcium ionophore or 5 [micro]M of either [Pam.sub.2][CysK.sub.4], CL264, [Pam.sub.2][CysK.sub.4] plus CL264, CL401, CL413, or CL531 for 5 min at 37[degrees]C and 5% C[O.sub.2].
Mechanistically, compared to unstimulated controls, stimulation of BMCMCs with 5 [micro]M [Pam.sub.2][CysK.sub.4], [Pam.sub.2] [CysK.sub.4] + CL264, or CL413, but not with CL264, CL401, or CL531, resulted in increased levels of ERK1/2 phosphorylation (Figure 3(d)), consistent with the induction of IL-6 secretion from BMCMCs by these ligands (Figure 3(b)).
Finally, because of the promising results obtained for the immune-activating capacities of CL531, in combination with the lack of ERK1/2 phosphorylation observed in BMCMCs stimulated with CL531, we decided to investigate whether CL531 might suppress allergic sensitization in a prophylactic vaccination approach using an established mouse model of OVA-induced intestinal allergy (Figure 5(a), for more information on the experimental groups see Repository Figure 5) [22].