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. 2011 Oct 5;31(40):14296–14307. doi: 10.1523/JNEUROSCI.2890-11.2011

Figure 3.

Figure 3.

Mutations of Elk-1 DEF or DEJL domain alter glutamate-induced H3 (Ser10) phosphorylation but preserve MSK-1 activation in vitro. A, Schematic representation of the Elk-1 mutants encoded by the plasmids used for transfections. Striatal neurons were transfected at DIV6 with cDNAs encoding HA-tagged versions of WT Elk-1 (Elk-1 Wild-Type), Elk-1 carrying a deletion of the DEJL domain (Elk-1 DEJL deletion), or a mutation of the DEF domain (Elk-1 DEF mutation). Twenty-four hours later, neurons were incubated in the absence (Control) or presence of glutamate (Glutamate; 10 μm) for 20 min, with or without U0126 (10 μm) applied 30 min before and during glutamate (Glutamate + U0126) application. B–D, Representative pictures of striatal neurons transfected with the WT or HA-tagged DEF Elk-1 mutants treated or not with glutamate in the absence or presence of U0126. Transfected cells were detected with anti-HA antibody (red) or P-Elk-1 with a phospho-specific antibody (green). White arrows indicate transfected striatal neurons that are P-Elk-1 positive. E–H, Quantifications of P-ERK1/2 (E), P-MSK-1 (F), P-Elk-1 (G), and P-H3 (Ser10) (H) immunoreactive cells, in HA-transfected cells. Note the strong decrease in P-Elk-1 and P-H3 (Ser10) signals in cells transfected with the DEF mutant, whereas the DEJL mutant only affects P-H3 (Ser10). Note also the total inhibition of all markers in the case of a pretreatment with U0126. E–H, Data (means ± SEM; n = 3–4 independent experiments per group) were analyzed using two-way ANOVA: for P-ERK1/2, effect of treatment, F(2,18) = 95.28, p < 0.001; effect of mutation, F(2,18) = 0.059, NS; for P-MSK-1: effect of treatment, F(2,18) = 53.71, p < 0.001; effect of mutation, F(2,18) = 0.39, NS; for P-Elk-1: effect of treatment, F(2,27) = 120.4, p < 0.001; effect of mutation, F(2,27) = 29.54, p < 0.001; for P-H3 (Ser10): effect of treatment, F(2,18) = 19.05, p < 0.001; effect of mutation, F(2,18) = 7.36, p < 0.01, followed by post hoc comparisons (Bonferroni's test). **p < 0.01, ***p < 0.001, control (Ctrl) versus glutamate (Glu); +p < 0.05, +++p < 0.001, glutamate versus glutamate + U0126; °p < 0.05, °°°p < 0.001, wild-type Elk-1 versus Elk-1 construct (DEF mutation or DEJL deletion).