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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: Nat Neurosci. 2012 Jul 22;15(9):1227–1235. doi: 10.1038/nn.3178

Figure 2. Aβ Oligomers Activate Fyn Kinase.

Figure 2

a Mouse E17 WT cortical neurons at 21 DIV were treated with 0–3 µM Aβo (monomer equivalent concentration, estimated Aβo 0–30 nM) for 20 min in the absence (left panel) or presence (right panel) of 6D11 pre-incubation for 1 h. Whole cell lysates were analyzed by anti-phospho-SFK (Tyr 416) or anti-Fyn immunoblot. Actin served as a loading control.

b Cortical neurons from E17 Prnp−/− mice after 21 DIV were treated with 0–3 µM Aβo for 15 min. Lysates were analyzed by anti-phospho-SFK (Tyr 416) or anti-Fyn immunoblot. GAPDH served as a loading control.

c Quantification of phospho-SFK level in the lysate, normalized to Fyn immunoreactivity. WT, n = 4; Prnp−/−, n = 4. Mean ± s.e.m. **, P < 0.001; one-way ANOVA (F=8.83; df=11), with Tukey post-hoc pairwise comparisons. Data normalized to 0 µM; the phospho-SFK densities without normalization at 0 µM for Aβo, Aβo+6D11, Aβm (Aβ monomer) and Aβo+Prnp−/− are 0.82±0.22, 0.86±0.10, 0.99±0.21 and 0.94±0.02.

d Cortical neurons from WT or Prnp−/− mice after 21 DIV were treated with 5 nM reelin for 25 min. Lysates analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, or anti-PrPC immunoblot.

e Quantification of phospho-SFK level in the lysates (from d) normalized to Fyn immunoreactivity from 3 biologically independent experiments. Mean ± s.e.m. *, P < 0.05; ***, P < 0.001; Student’s two-tailed t test.

f Cortical neurons from WT after 21 DIV were treated with 0–1 µM Aβo. Prior to Aβo exposure, the indicated cultures were pre-treated with 5 mg/ml methyl-β-cyclodextrin (MBCD) for 1 h or 0.1 unit of PI-PLC for 10 min. Whole cell lysates were analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, or anti-PrPC immunoblot. Actin served as a loading control.

g Quantification of phospho-SFK level in the lysates (from f) normalized to Fyn immunoreactivity from 3 independent experiments. Mean ± s.e.m. *, P < 0.05; Student’s two-tailed t test.

h Cortical neurons from WT after 21 DIV were treated for with 1 µM Aβo, 10 µg/ml anti-PrPC antibodies (6D11 or SAF32) or control IgG (Con) antibodies, followed by adding 5 µg/ml mouse anti-IgG antibodies for crosslinking (α-IgG). Whole cell lysates were analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, or anti-PrPC immunoblot. Actin served as a loading control.

i Quantification of phospho-SFK level normalized to Fyn immunoreactivity from 3 independent experiments, as in h. Mean ± s.e.m. ***, P < 0.001; *, P < 0.05; one-way ANOVA (F=5.38; df=7), Tukey post-hoc comparisons.