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Fig. 2. Agrin inhibits the neurite extension of cultured Xenopus spinal
neurons. (A) Microscopic time-lapse images of a neurite at various time points
after the addition of agrin. Top panel, treated with BSA (50 µg/ml in bath)
as control; bottom panel, treated with agrin (20 ng/ml in bath). Broken lines
indicate the duration of treatment. (B) Normalized neurite extension rate of
neurons treated with agrin compared with that of control, BSA. The neurite
extension rate was measured and normalized by comparing the neurite extension
rate before and after agrin treatment. Each value represents the
average±s.e.m.; *P<0.01 (Student's
t-test). (C) A dose-response curve of the effect of agrin on neurite
extension. Negative values represent the average length of neurite retraction.
Approximately 20-40 neurons were assayed for each concentration of agrin
tested (0.1-50 ng/ml). (D) The inhibitory effect of agrin on neurite extension
was abolished by an agrin-neutralizing antibody (Agrin+Ab), or by
overexpressing a MuSK mutant that comprised the extracellular domain of MuSK
fused with the Fc region of an Ig (EC-MuSK). Microscopic time-lapse images of
a neurite extending from a neuron under different conditions. Cultured spinal
neurons prepared from Xenopus embryos were injected with empty vector
(Mock) or an MuSK mutant (EC-MuSK), and treated with agrin (20 ng/ml). For
treatment with agrin+Ab, agrin was preincubated with its neutralizing antibody
(40 ng/ml) at 4°C overnight. (E) Histogram showing the normalized average
neurite extension rate under different conditions, as in D. The neurite
extension rate was normalized by comparing neurite extension rate before and
after the application of agrin. Each value represents the
average±s.e.m.; *P<0.01 (Student's
t-test).
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