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. 2014 Jul 15;12(7):e1001908. doi: 10.1371/journal.pbio.1001908

Figure 1. Gephyrin is palmitoylated in vivo.

Figure 1

(a) Mouse brain lysates were fractionated via ultracentrifugation and immunoblotted with gephyrin and PSD-95–specific antibodies. (b) Representative immunoblots of enriched synaptosomes after extraction with Triton X-100 or Saponin, overlayed with a discontinuous sucrose gradient and ultracentrifugation. Ten fractions were taken from top to bottom. (c) Gephyrin palmitoylation detected by metabolic labeling with 17-ODYA followed by click chemistry and affinity purification. (d) Basal palmitoylation of gephyrin in primary hippocampal neurons demonstrated by the ABE assay. Replacing hydroxylamine (HA) with a Tris-buffer (−HA) served as an internal negative control to proof efficient blockade of thiols. Palmitoylation of gephyrin was blocked with 50 µM 2-BP for 14 h. (e) Gephyrin palmitoylation status in the cytosolic (S2) compared to synaptosomal (P2) fraction analyzed by the ABE assay. Lanes of the top panel have been spliced from the same membrane. Volumes of the S2/P2 fractions have been adjusted to similar amounts of gephyrin (right panel). Experiment was performed with two individual synaptosomal preparations with essentially the same results.