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. Author manuscript; available in PMC: 2014 Nov 13.
Published in final edited form as: Nat Commun. 2014 Oct 9;5:5066. doi: 10.1038/ncomms6066

Figure 2. Pin1 negatively modulates NL2/gephyrin interaction.

Figure 2

(a) Representative IP of FLAG epitopes from samples of HEK293 cells co-expressing gephyrin-FLAG and NL2HA and treated for 48h with PiB 2.5μM, DMSO (mock) or untreated. IP was also performed on NL2HA single transfected cells as a negative control. Nitrocellulose membranes were probed with anti-HA and anti-FLAG antibodies. The histogram on the right shows the relative amount of NL2 coprecipitated by gephyrin-FLAG in control and PiB treated cells obtained from densitometric analysis (n=5, mean values ± s.d., **P < 0.001, Student’s t-test). (b) Lysates of HEK cells transfected with gephyrin-FLAG in the presence of NL2HA or NL2HAS714A or with gephyrin alone (as a negative control) were immunoprecipitated with anti-HA agarose. Immunoprecipitates were analysed by Western blotting using anti-FLAG and anti-HA monoclonal antibodies. Arrowhead indicates the IgG heavy chains. The histogram on the right shows the relative amount of gephyrin-FLAG in complex with either NL2HA or NL2HAS714A coprecipitated by anti-HA agarose obtained from densitometric analysis (n=5, mean values ± s.d., **P < 0.001, Student’s t-test ). (c) CoIP of endogenous NL2/Pin1 complexes from DSP cross-linked brain homogenates of Pin1+/+ or Pin1−/− mice. Western blots were performed with anti-NL2 polyclonal and anti-Pin1 monoclonal antibodies. Rabbit IgGs were used as negative control. An increased amount of gephyrin co-precipitates in complex with NL2 in the absence of Pin1 expression. Arrowhead indicates the IgG heavy chains. The histogram on the right shows the relative amount (obtained from densitometric analysis) of endogenous gephyrin coprecipitated by endogenous NL2 from both mouse genotypes (n=8, mean values ± s.d., *P < 0.01, Student’s t-test ). (d) A similar experiment described in c was carries out on hippocampus isolated from of Pin1+/+ or Pin1−/− mice. The histogram on the right shows the relative amount (obtained from densitometric analysis) of endogenous gephyrin coprecipitated by endogenous NL2 from both mouse genotypes (n=4, mean values ± s.d., **P < 0.001, Student’s t-test ). AU arbitrary units. Full images of western blots are in Supplementary Fig.5.