Figure 4.

CaMKII-dependent phosphorylation is necessary for STH. A, Western blot showing the effect of pan-neuronal overexpression of CaMKII-inhibitory peptide ala on synapsin phosphorylation. Inhibition of CaMKII activity causes a significantly increased abundance of a higher electrophoretic mobility synapsin isoform (left blot) that is not recognized by the α-Psyn-S6 antibody. In turn, the abundance of an S6-phosphorylated isoform is reduced (right blot). Western blot signals of the loading control synapse-associated protein (SAP47) remain unchanged across the indicated genotypes. Flies with adult-specific (B) overexpression of ala peptide (LN1-Gal4; tub-Gal80^ts> UAS-CaMKII-ala) (F_(1,43) = 26.182, p < 0.001, two-way ANOVA; Turkey test for 18°C flies, q = 10.066, p < 0.001 and for 29°C-treated flies, q = 0.167, p = 0.907) and (C) knock down of CaMKII (LN1-Gal4; tub-Gal80^ts> UAS-CaMKII^RNAi) (F_(1,35) = 80.498, p < 0.001, two-way ANOVA; Turkey test for 18°C flies, q = 17.442, p < 0.001 and for 29°C flies, q = 0.503, p = 0.725) in AL LN1 neurons fail to show STH at 29°C, whereas flies maintained at the permissive temperature (18°C) displayed normal STH. D, Histogram showing the effect of conditional overexpression of CaMKII inhibitory peptide in LN1 neurons (LN1-Gal4; tub-Gal80^ts> UAS-CaMKII-ala), on LTH (F_(1,28) = 3.402, p = 0.077, two-way ANOVA; Turkey test for 18°C flies, q = 4.523, p = 0.004 and for 29°C flies, q = 0.557, p = 0.697). Light gray and dark gray bars correspond to naive or mock-exposed and post-odor exposure responses, respectively, measured for 10⁻³ dilution of EB. Temperature-shift protocols for adult-specific CaMKII inhibition and knock down experiments are same as the schematic shown in the Figure 2D. Error bars indicate ± SEM. ***p < 0.001 and **p < 0.01 equal to 0.001 (Student's t test with Bonferroni correction). Refer to Tables 1 and 2 for N.