Figure 4

CaMKII activation of R192Q KI trigeminal neurons is reversed by ω-agatoxin. A, Microphotographs of immunofluorescence experiments with anti-phospho Thr286 CaMKII antibody (recognizing the active form of CaMKII) of WT and KI trigeminal ganglia (left panel) or trigeminal neurons in culture (right panel). Bar = 50 μ m. B, Example of western immunoblots of total protein lysate from WT and KI trigeminal neurons in culture using antibodies recognizing the active form of CaMKII (phosphorylated Thr286, upper lanes) or anti-total CaMKII antibody (middle lanes). Equal loading was tested with β-tubulinIII antibodies (bottom lanes). p = 0.03, (n = 4). CaMKII activation is prevented by treatment with ω-agatoxin (200 nM, 24 h). Histograms (right) demonstrate significant increase in active (threonine-phosphorylated) CaMKII in KI neurons, an effect prevented by ω-agatoxin (n = 4, #p = 0.027). C, Western immunoblot experiments of trigeminal ganglia from WT and KI mice, using the same anti-phospho-CREB antibody (43 kDa). Gel loading quantification is obtained using anti-β-tubulinIII antibody. Histograms (right) show no significant difference in CREB phosphorylation between the two conditions. n = 3, p > 0.05.