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. 2010 Apr 21;107(19):8830–8835. doi: 10.1073/pnas.0910644107

Fig. 6.

Fig. 6.

Postsynaptic eB3 acts through Erk1/2 to control synapse density. (A) Various purification fractions from three P30 mouse brains. Blots are probed with antibodies recognizing eB3, NR1, GluR2, PSD-95, and Erk1/2 (n = 3). (B) EB3 immunoprecipitated from synaptosomes and resulting Western blots were probed with anti-Erk1/2 and anti-eB3 antibodies (n = 3). Lysates from the same preparations are shown in lower blots. (C) GST fusion proteins of wt (eB3intra) or L293A mutant (eB3 L293A) intracellular domains of eB3 were expressed in bacteria and used in GST pull-down assay with E19 rat brain lysates. Resulting elutates and input to the columns were analyzed by Western blotting with anti-Erk1/2 antibody. Fusion protein expression was analyzed on a Western blot with anti-GST antibody. (D) Quantification of relative Erk1 and Erk2 pull down as compared with total Erk1 and Erk2 input (n = 3). (E–G) Density of excitatory synapses in rat cortical neurons transfected with DN-MEK and either control or eB3shRNA constructs at DIV 10. (E) Quantification of the frequency of mEPSCs vector control as in Fig. 2, DN-MEK (n = 11), CA-MEK (n = 7), eB3 shRNA#1 plus DN-MEK (n = 12), eB3 shRNA#1 plus CA-MEK (n = 6). (F) Examples of staining for markers PSD-95 (red) and VGlut (blue) in transfected neurons. (G) Quantification of the synapse density. Vector control (n = 27), eB3 shRNA#1 (n = 39, P = 0.1212). (H) EB3−/− and wt neurons transfected with GFP alone or GFP and DN-MEK cultured on wt mouse cortical neurons. GFP (green), PSD-95 (red), and vGlut1 (blue). Arrowheads indicate colocalized PSD-95 (red) and vGlut (blue) puncta in transfected neurons. (I) Quantification of synaptic density in heterogenotypic cultures (wt/wt, n = 18; eB3−/−\wt, n = 30; eB3−/−\wt+DN-MEK, n = 34). (J) Neurons transfected with GFP and vector control or eB3 shRNA at DIV 0 [anti-GFP (green), anti-Erk1/2 (red), and anti-VGlut1 (blue)]. (Scale bar, 5 μm.) Arrowheads indicate VGlut1 puncta. Straight arrows indicate cytoplasmic Erk1/2; curved arrows indicate nuclear Erk1/2. (K) Quantification of percent nuclear Erk1/2 after transfection with vector control (n = 6 transfections) or eB3 shRNA (n = 6 transfections). A total of 50–300 cells were scored for each experiment. (L) Dendrites of control or eB3 shRNA–expressing neurons stained for GFP (green), Erk1/2 (red), and vGlut1 (blue). Arrowheads indicate VGlut puncta in transfected neurons. (M) Quantification of ratio of synaptic Erk to dendritic Erk staining in control and eB3 shRNA-expressing neurons. Vector control (n = 22), eB3 shRNA #1 (n = 24). (J–M) Experiments were conducted following a 1-h treatment with blockers of neuronal activity (1 μM TTX), NMDA receptors (50 μM APV), and L-type calcium channels (50 μM nifedipine). (Scale bars, 3 μm except in J.) *P < 0.03, **P < 0.01, ***P < 0.0001. Error bars indicate SEM.