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. 2014 Oct 9;5:5066. doi: 10.1038/ncomms6066

Figure 1. NL2 is a proline-directed substrate.

Figure 1

(a) Amino acid sequence of the NL2 CD. In bold is marked the unique Pin1 consensus motif (S714-P). The gephyrin-binding domain and the proline-rich region are highlighted in bold. (b) Representative immunoblotting of endogenous NL2 immunoprecipitated (IP) from mouse brain and probed with the anti-MPM2 that specifically recognizes phosphorylated S/T-P motifs and anti-NL2. Rabbit IgGs were used as negative control (IgG) (n=4). (c) Representative immunoblotting of overexpressed NL2HA lacking the gephyrin binding domain (NL2HA-ΔGBD) and the corresponding point mutant (NL2HA-ΔGBDSer714Ala) immunoprecipitated by the phospho-specific MPM2 antibody. Western blot analysis was carried out with anti-HA monoclonal antibody. Mouse IgGs were used as negative control (n=5). (d) Co-immunoprecipitation (Co-IP) of endogenous NL2 and Pin1 from DSP cross-linked brain homogenates of Pin1+/+ or Pin1−/− mice. Western blots were performed with anti-NL2 polyclonal and anti-Pin1 monoclonal antibodies. Mouse IgGs were used as negative control. Asterisk indicate the IgG light chains (n=6). (e) FLAG epitopes from cross-linked samples of HEK293 cells co-expressing Pin1-FLAG and NL2HA-ΔGBD or NL2HA-ΔGBDS714 were immunoprecipitated by anti-FLAG antibody. Western blot was performed with anti-HA and anti-FLAG monoclonal antibodies. Mouse IgGs were used as negative control (n=4). Full images of western blots are in Supplementary Fig. 5.