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. 2015 Feb 18;35(7):3155–3173. doi: 10.1523/JNEUROSCI.0586-14.2015

Figure 8.

Figure 8.

Chmp2b is part of a stable ESCRT-III-type complex. A, Rat forebrain homogenates were fractionated as described in Materials and Methods. Equivalent amounts of each fraction were analyzed by Western immunoblotting with the indicated antibodies. Both Chmp2b and Chmp4b were enriched in the PSD fraction. B, PSDI was re-extracted with either 0.5% Triton X-100 or 3% Sarkosyl to yield insoluble fractions PSDII or PSDIII, respectively. Equivalent amounts of each fraction were analyzed by Western immunoblotting. C, Synaptoneurosomes were solubilized with the indicated detergents and centrifuged at high speed. The resulting supernatants and pellets were analyzed by immunoblotting with the indicated antibodies. D, Gradient-purified synaptoneurosomes were lysed in 1% DOC, insoluble material was removed, and the extract was fractionated by size-exclusion chromatography on a Sephacryl S-300 column. Column fractions were analyzed by immunoblotting with the indicated antibodies. Apparent molecular weights (MW) were calibrated with protein markers. A subset of Chmp2b displayed the same high MW as the PSD-95 complex. E, Monomeric recombinant His-tagged Chmp2b was incubated with either 1% Triton X-100 or 1% DOC and fractionated by size-exclusion chromatography on a Sephacryl S-300 column. Fractions were analyzed by immunoblotting with an anti-His tag antibody. DOC did not cause detectable aggregation of monomeric Chmp2b. The elution peaks of synaptic Chmp2b-containing fractions resolved on the same column are indicated for reference. F, Fractions corresponding to HMW and LMW Chmp2b populations were separately pooled and immunoprecipitated with anti-Chmp2b antibody or control IgG. The immunoprecipitates were analyzed by immunoblotting with anti-Chmp2b or anti-Chmp4b antibody. Chmp4b coprecipitated with Chmp2b in the HMW peak. G, Synaptoneurosome extracts were prepared as in D and immunoprecipitated with anti-Chmp2b antibody or control IgG. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies.