Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses
Figure 3
Palmitoylation on Cys212 and Cys284 is essential for gephyrin localization and clustering in neurons.
(a) Scheme of gephyrin domain architecture with surface-exposed and Palm-CSS 3.0–predicted cysteine residues. (b) Immunoprecipitation of ABE-assay–processed gephyrin cysteine-to-serine mutants expressed in HEK293 cells. Streptavidin-HRP shows palmitoylation status of individual variants, whereas GFP-immunoblot was used as the loading control. Experiment was repeated three times with similar results. (c) SDS-PAGE of ABE-assay–processed and Ni-NTA affinity-purified gephyrin and LC-MS/MS analysis of the protein bands. (d) Quantitative comparison of cluster size of GFP-tagged WT and mutant gephyrin variants in primary hippocampal cultures. (e) Total intensity of WT gephyrin or 212,284Geph clusters (f) Representative dendrites of gephyrin-GFP and 212,284Geph. Scale bar, 5 µm. All quantifications are means ± SEM (***p<0.001 using Student's t test; NS, not significant). At least three independent cultures were used per experiment.