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Fig. 1. Characterization of neurospheres. (A-C) Immunocytochemistry
identified nestin (A), GFAP (B), S-100ß (C), ßIII tubulin
(D), and the ecto-nucleotidases NTPDase2 (E) and TNAP (F)
in all neurospheres investigated (representative images). Scale bars: 50
µm. (G) Analysis of ecto-phosphatase activity. Viable neurospheres
were incubated in the presence 1 mM ATP, ADP, AMP, PNPP or PNP-TMP. To
determine the contribution of TNAP activity, ATP hydrolysis by neurospheres
was analyzed in the presence of 1 mM levamisole. No phosphodiesterase activity
(PNP-TMP hydrolysis) was observed. Phosphatase activities were normalized to
the activity obtained with ATP as a substrate. The 100% value corresponds to
3.2±1.1 nmoles Pi/min/100 neurospheres (mean±s.d.,
n=9-12, *P<0.05,
**P<0.01). (H) RT-PCR products revealing the
presence of mRNAs in neurosphere extracts encoding NTPDase2 and TNAP.
(I) Immunoblot using total neurosphere protein (5 µg/lane) detecting
protein bands of 70 and 80 kDa (arrowheads) corresponding to NTPDase2 and
TNAP, respectively.
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