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Fig. 3. Expression of WT and mutant human profilin I in PC12 cells: a quantitative analysis of neuronal differentiation. (A) Western blot showing the expression levels of human WT and mutant profilins in the stably transfected PC12 cells. The levels were quantified by densitometry of the blot. The apparent higher molecular weight of the profilin IW3A mutant is also observed for the purified recombinant protein and is probably due to the introduced substitution (data not shown). (B) Representative images of the cell lines used in this study. The cells were grown on collagen-coated plastic and stimulated with NGF/forskolin for 24 hours. The profilin IR136D-expressing cells are visibly more differentiated. Bars, 20 µm. (C) Percentage of differentiated cells at 24 and 96 hours after NGF/forskolin stimulation. Results of two independent experiments are presented as means ± s.e.m.; n=317-355 cells. (D) Percentage of differentiated cells with two or more neurites at different time points after the addition of NGF/forskolin. Results of three independent experiments are presented as means ± s.e.m.; n=355-404 cells. (E) Percentage of neurites with branches, 96 hours after the addition of NGF/forskolin. n ranges from 136 to 159 cells, covering two independent experiments. (F) Profilin IR136D and profilin IW3A cells have longer neurites and profilin IR74E cells have shorter neurites from profilin IWT. The length of the longest neurite per cell (n=50 cells) was measured 96 hours after the start of differentiation and these lengths were plotted from short to long. The neurites can be divided into two populations: short – newly formed neurites up to 80 µm; and long – established neurites longer than 80 µm. The difference between the cell lines is most obvious in the population of longer neurites. 
, profilin IWT; 
, profilin IW3A; 
, profilin IR74E; 
, profilin IR136D.
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