Fig. 4. Time course of receptor phosphorylation. (A and B) The amount of 33P incorporated into proteins in response to various incubation times with 100 nM NT was quantified after immunoprecipitation of the WT HA-tagged-mNTR2 and the HA-Y237A-mNTR2 mutant. (A) Autoradiography and western Blot (WB) of phosphorylated WT HA-mNTR2 (lanes a-c) and HA-Y237A-mNTR2 mutant (lanes d-f) receptors from a representative experiment. (B) Summary of the quantification of receptor phosphorylation. Values for each receptor were expressed in arbitrary unit (basal intensity of phosphorylation in the absence of NT per pmol of receptor) and are means±s.e.m. of three independent experiments. *P<0.05. (C,D) The tyrosine phosphorylation of the WT GFP-tagged-mNTR2 and the GFP-Y237A-mNTR2 mutant upon NT treatment was assessed by phosphotyrosine immunoprecipitation. Cells transfected with the WT GFP-tagged-mNTR2 (C) or the GFP-Y237A-mNTR2 mutant (D) were incubated with 100 nM NT for indicated times in the absence or in the presence of 100 µM genistein, a tyrosine kinase inhibitor. Immunoprecipitation (IP) was performed on total cell extracts using mouse anti-phophotyrosine antibodies. Western blot (WB) were probed with rabbit anti-GFP antibodies. The western blot shown is representative of two independent experiments.