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Fig. 4. Membrane attachment of the ALK intracellular domain is required to induce neurite extension of PC12 cells and MAP kinase activation. (A) PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for 2 days. Then cells were fixed, permeabilized and immunofluorescence assay was performed. Cells expressing the ALK-FH-derived proteins were labeled with the anti-HA-tag antibody (12CA5) and cell nuclei were colored with the nucleic acid stain Hoechst 33258. Bar, 100 µm. (B) Quantification of the effect of the induced activation of the different proteins on neurite outgrowth, as indicated in the Materials and Methods. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Time course of ERK 1/2 phosphorylation following dimerizer treatment. PC12 cells expressing the indicated proteins were cultured overnight in a 1% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-HA-tag or the anti-P-ERK antibody and then reprobed with the anti-PY (4G10) or the anti-ERK antibody.
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