Dynamics of PLCγ and Src Family Kinase 1 Interactions during Nuclear Envelope Formation Revealed by FRET-FLIM
Figure 6
PLCγ Y783 phosphorylation requires SFK catalytic activity and GTP hydrolysis.
(A) Experiment was performed as in Figure. 5 with nuclei treated for 5 minutes with 1 mM GTP or 2 mM GTPγS. Nuclei were also pre-incubated with the Src inhibitor PP2 or its inactive analogue PP3 (both 10 µM). After this time nuclei were fixed and stained with Hoechst (blue), DiOC6 (green) and anti-pY783 (red), and imaged by confocal microscopy. (B) Nuclei in A were scored for multiple pY783-positive sites on the nucleus surface. Data are expressed as mean+s.e.m from three independent experiments. Scale bar is 1 µm. (C) Experiments performed as in (B) with NE proteins subjected to an anti-pY783 western blot. pY783 bands were normalised to total PLCγ and expressed as a fold-change compared to nuclei assembled in the presence of ATP only. Data are expressed as mean+s.e.m from at least three independent experiments.