Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses
Figure 2
Palmitoylation of gephyrin is essential for clustering at GABAergic synapses.
(a) Immunostaining of gephyrin (red) and VGAT (green) in primary hippocampal neurons. Insets are taken from 20 µm dendritic segments. Arrows point to nonsynaptic gephyrin clusters upon incubation with 2-BP. Scale bar, 20 µm. (b–d) Histograms showing quantitative comparisons of (b) gephyrin colocalization with VGAT, (c) gephyrin cluster size, and (d) VGAT puncta area in control and 2-BP–incubated cultures. (e) Quantification of percentage of palmitoylated proteins in mouse brains. Immunoblots with increasing volumes of ABE-assay–processed protein lysates were used to create a band-intensity standard curve for ABE-assay eluates of gephyrin and PSD-95. The experiment has been performed with three brains. (f′) Streptavidin-HRP immunoblot served as a specificity and accuracy control of the assay. L, lysate; +HA, samples processed with hydroxylamine; +HAB, +HA supernatant after Neutravidin beads sedimentation to prove efficient affinity-purification of biotinoylated proteins; −HA, internal control of the ABE assay without hydroxylamine.