Fig. 2. Induced kinase activation of the full-length receptor ALK results in neurite extension and ERK 1/2 activation. (A) Dimerizer dose-dependent effect on neurite outgrowth induced by the activation of ALK-FH protein. PC12 cells were electroporated with the ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated without or with increasing concentrations of dimerizer as indicated for 2 days. Then immunofluorescence assay was performed using the anti-HA-tag antibody (12CA5); 100 transfected cells were counted and cells bearing neurites longer than twice the diameter of the cell body were scored as differentiated. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (B) Neurite outgrowth induced by ALK-FH dimerization is a result of its own kinase activity. PC12 cells were electroporated with the indicated constructs (25 µg) and then submitted to the same protocol as described in (A). Cells were treated with a dimerizer concentration of 20 nM. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Time course of (C) ALK-FH and (D) ERK 1/2 phosphorylation following dimerizer treatment. ALK-FH-transfected PC12 cells were incubated with the dimerizer (20 nM) for the indicated periods. As controls, non-transfected cells (Mock) or cells transfected with the dALK-FH mutant were treated with the dimerizer during 30 minutes. Cells were lysed in a RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-PY (4G10) or the anti-P-ERK 1/2 antibody (upper panels) and then reprobed with the indicated antibodies (lower panels). The P-ERK 2 densitometry quantification was performed using the Odyssey Imaging System on three independent experiments. Statistical analyses were carried out by Student's t-test (***P<0.005). Asterisk in C indicates a 160 kDa protein that was not revealed by the anti-HA-tag antibody and therefore not related to ALK.