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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 3. Impact of gephyrin S270A and S319A in NL2/gephyrin interaction.

Figure 3

(a) GST-NL2-CD pull-down from samples of HEK293 expressing EGFP-gephyrin full-length (FL), EGFP- gephyrin 310-736 (E-310), EGFP-gephyrin 326-736 (E-326) and EGFP-gephyrin GC. GST was used as negative control. Pulled down eGFP-gephyrin variants were detected using an anti-GFP monoclonal antibody. The bottom panels show the levels of GST and GST–NL2CD in the pull-down assays (Ponceau staining) (n=8). (b) EGFP-gephyrin Δ319 to 329 was tested in similar pull-down assays. Western blots in (a) and (b) were performed using anti-GFP antibody. Gephyrin requires amino acid sequence 319-329 for its efficient recruitment by NL2 (n=6). (c) Representative IP of HA epitopes from samples of HEK293 cells co-expressing NL2HA and EGFP-gephyrin wild type, EGFP-gephyrinS270A or EGFP-gephyrinS319A. Nitrocellulose membranes were probed with anti-HA and anti-GFP antibodies. EGFP-gephyrin single transfected cells incubated with HA-agarose were used as negative controls. The histogram on the right shows the relative amount of eGFP-gephyrinWT and point mutants coprecipitated by NL2HA (n=4, mean values ± s.d., P > 0.05). (d) Representative images of hippocampal neurons transfected with EGFP-gephyrin and EGFP-gephyrinS270A point mutant immunolabeled for endogenous NL2 (magenta) and VGAT (blue) at DIV10. Enlarged boxed areas are shown aside to the corresponding full view image. Post-synaptic clustering is demonstrated by apposition of gephyrin/NL2 clusters to VGAT positive terminals on the merge window. Scale bars: 20μm in full view images and 5μm in enlarged panels. (e) Distribution histograms of the % of gephyrin clusters colabeled with NL2 (79 ± 5% in EGFP-gephyrinWT vs 77 ± 4% in EGFP-gephyrinS270A), % of NL2 clusters colabeled with gephyrin (48 ± 5% in EGFP-gephyrinWT vs 71 ± 4% in EGFP-gephyrinS270A), % of NL2 synaptically localized (29 ± 2% in EGFP-gephyrinWT vs 43 ± 6% in EGFP-gephyrinS270A) and NL2 clusters intensity (119 ± 15 AU in EGFP-gephyrinWT vs 102AU ± 6 in EGFP-gephyrinS270A). The number of transfected hippocampal neurons investigated in each experiments (4 independent experiments) were as follow: n=15 for eGFP-gephyrinWT, n=10 for eGFP-gepyrinS270A (for each neurons at least 4 dendritic ROIs were measured, mean values ± s.d., *P < 0.01, Student’s t-test). AU: Arbitrary Units.