Skip to main content
. 2007 Apr 26;404(Pt 1):31–43. doi: 10.1042/BJ20061483

Figure 3. Regulation of Ng binding to PA.

Figure 3

(A) Ng binding to PA was analysed in protein–lipid overlays as described in the text, except that Ng was incubated previously with different amounts of CaM for 30 min at 25 °C before the mixture was added to egg-yolk PA-spotted strips. (B) Recombinant Ng was phosphorylated by PKC and separated from non-phosphorylated Ng by affinity chromatography in CaM–agarose columns. Binding of Ng (●) and phospho-Ng (■) was assayed as described in the text. (C) Binding of biotin–IQ peptide (●), corresponding to Ng-(29–47), and its phosphorylated form (biotin–IQ-P) (■) to egg-yolk PA-spotted strips was analysed using the method described for protein–lipid overlay assays, except that the primary and secondary antibodies were replaced by an incubation with HRP-labelled streptavidin (1:2000 dilution) for 1 h. Results in (A), (B) and (C) are means±S.E.M. for at least three independent experiments. (D) To analyse CaM competition, 5 μg of CaM was added to liposomes of the indicated composition along with Ng to the binding mixture. Typical Western blots representative of three independent experiments are shown. S, supernatant; P, pellet.