(A) E18 rat cortical neurons were stimulated with clustered ephrinB2 (efnB2), ephrinB1 (efnB1) or Fc control for 3, 10, 30 or 60 mins, and then lysates were immmunoblotted with a general anti-phospho-Eph antibody (Shamah et al, 2001) or a loading control anti-phospho-cofilin antibody.
(B) Growth cone morphology of Fc-control (top) and EphrinB2 (bottom) stimulation conditions. Uncollapsed (yellow arrows) and collapsed (white arrows) growth cones were visualized with phalloidin (Oregon-green).
(C) Time-course analysis of cortical (E18 + 2 DIV) neuron growth cone collapse upon preclustered Fc-control or ephrinB2/Fc addition (mean +/− SEM, n=9 from 3 independent experiments).
(D) Time-course analysis of cortical (E18 + 2 DIV) neuron growth cone collapse upon preclustered Fc-control or ephrinB1/Fc addition (mean +/− SEM, n=9 from 3 independent experiments).
(E) Cortical GCC (E18 + 2DIV) after 1 hr treatment with indicated preclustered ephrins or Fc control (mean +/− SEM, n=3, ***p<0.001 ephrin/B2 vs. Fc control, Student’s t-test).