Figure 1. dAlk protein is an active RTK expressed in the adult CNS.

(A) Frontal paraffin sections stained with an anti-dAlk antibody, demonstrate accumulation of the receptor in neuropil areas, especially in the Calyx (Cx) (1) the protocerebral bridge (pb) (2) the suboesophageal ganglion (sog) (3,4) and antennal lobe (al) (4) marked with black arrowheads. dAlk is absent from the α, β, and γ lobes of the mushroom bodies and pedunculus (p; white arrowheads) (3,4). Bar = 50 µm. (B) A representative semi-quantitative immunoblot and its quantification illustrate significant reduction of endogenous dAlk upon RNAi-mediated abrogation using two different UAS-Alk^RNAi transgenes (UAS-Alk^RNAi and UAS-Alk^RNAiKK) when driven with Elav-Gal4 . ANOVA indicated significant effects of genotype (F_(2,15) = 6.51, p<0.01, n = 5). Student's t-test revealed significant differences between controls and the two different UAS-Alk^RNAi lines (p<0.004 and p<0.02 for UAS-Alk^RNAi and UAS-Alk^RNAiKK, respectively). Tubulin levels served as loading control. (C) Immunohistochemical demonstration of anti-dAlk antibody specificity in adult brain. Representative 40 µm confocal z-stacks from brains of adult w¹¹¹⁸ (upper panel) and flies expressing the UAS-Alk^RNAi transgene under control of the MB-specific c772-Gal4 driver stained with the anti-dAlk antibody. dAlk accumulation within the dendrites (black arrowhead) of wild type flies was highly reduced in the dendrites of flies expressing the UAS-Alk^RNAi transgene. White arrowheads point to the protocerebral bridge where the Gal4 driver is not expressed, showing equal levels of staining between control and experimental flies. Confocal images were acquired within the same range and using the same acquisition settings. They were then converted to grayscale and inverted. Scale bar = 50 µm. (D) Manipulation of dAlk activity in Ras2-expressing neurons affects ERK phosphorylation levels. Representative semi-quantitative immunoblot (left) and its quantification (right) showing alteration of ERK phosphorylation levels upon expression of dAlk transgenes in the Ras2-expressing cells. Levels of pERK were significantly higher upon over-expression of UAS-Alk^WT , UAS-Jeb and UAS-Alk^CA relative to w¹¹¹⁸ control (p = 0.04, p = 0.0053, p = 0.03 respectively, n = 3, Student's t-test). Accordingly, pERK levels were significantly lower upon expression of UAS-Alk^DN or UAS-Alk^RNAi transgenes relative to w¹¹¹⁸ control (p = 0.0002 and p = 0.01, n = 3, Student's t-test). The amount of total-ERK protein is not affected. Error bars denote S.E.M.