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. 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 pulldown 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 eGFPgephyrin variants were detected using an anti-GFP monoclonal antibody. The bottom panels show the levels of GST and GST-NL2-CD in the pulldown assays (Ponceau staining) (n=8). (b) EGFP-gephyrin Δ319 to 329 was tested in similar pulldown 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 WT, 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 co-precipitated 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, 20 μm in full view images and 5 μm in enlarged panels. (e) Distribution histograms of the % of gephyrin clusters colabeled with NL2 (79±5% in EGFP-gephyrinWT versus 77±4% in EGFP-gephyrinS270A), % of NL2 clusters colabeled with gephyrin (48±5% in EGFP-gephyrinWT versus 71±4% in EGFP-gephyrinS270A), % of NL2 synaptically localized (29±2% in EGFP-gephyrinWT versus 43±6% in EGFP-gephyrinS270A) and NL2 clusters intensity (119±15 a.u. in EGFP-gephyrinWT versus 102 a.u.±6 in EGFP-gephyrinS270A). The number of transfected hippocampal neurons investigated in each experiments (four independent experiments) were as follow: n=15 for eGFP-gephyrinWT, n=10 for eGFP-gepyrinS270A (for each neurons at least 4 dendritic regions of interests were measured, mean values±s.d., *P<0.01, Student’s t-test).