Journal: Febs Letters

Article Title: The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src

doi: 10.1016/j.febslet.2015.05.033

Figure Lengend Snippet: N-Srcs have enhanced levels of Y416 auto-phosphorylation in vitro and in cells compared to C-Src. (A) 200 nM HisΔ80C-, N1- or N2-Src was incubated at 30 °C with 0.5 mM ATP for 0, 1 or 3 h and processed for immunoblotting with anti-pY416 and anti-His (loading control). pY416 immunoreactivity was obtained by densitometry with ImageJ and normalised to the anti-His signal. Data are plotted as mean ± S.E.M., from n = 3 independent experiments. (B) B104 cells were transfected with plasmids encoding FLAG-tag alone (control) or C-terminal FLAG-tagged full length C-, N1- or N2-Src, and the lysed after 48 h. The lysates were analysed by immunoblotting with anti-FLAG (FLAG), anti-phosphotyrosine (pY), anti-pY416 (active Src), anti-pY527 (inactive Src) and β-actin (loading control). The immunoblots are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-Src pY527 and anti-Src pY416 were from Cell Signaling Technologies (Hitchin, UK).

Techniques: Phospho-proteomics, In Vitro, Incubation, Western Blot, Control, Transfection, FLAG-tag