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. Author manuscript; available in PMC: 2016 Mar 15.
Published in final edited form as: Biochem J. 2015 Mar 15;466(3):625–637. doi: 10.1042/BJ20141202

Figure 1. ESCRT-II binds directly to VPS-20 in solution.

Figure 1

(A) The specificity of antibodies directed against ESCRT-II was tested using C. elegans extracts generated from wild type animals and animals depleted of all three ESCRT-II subunits. The three bands corresponding to VPS-36, VPS-22, and VPS-25 are substantially diminished following RNA interference. Molecular mass markers are shown on the right, and an immunoblot directly against actin was conducted as a loading control. (B) Antibodies directed against all three subunits of the ESCRT-II complex were used to isolate ESCRT-II and any interacting partners from C. elegans embryo extracts and subjected to solution mass spectrometry analysis. Identified components of the ESCRT machinery, their percent sequence coverage, and their molecular masses are shown. (C and D) Antibodies directed against ESCRT-II, VPS-20, or STAM-1 were used to conduct immunoprecipitations from C. elegans embryo extracts. Recovered proteins were separated by SDS-PAGE and immunobloted using antibodies directed against ESCRT-II (panel C) or VPS-20 (panel D). Inputs for each immunoprecipitation are shown on the left. (E) Immunoprecipitations using ESCRT-II antibodies were conducted using a low speed supernatant (LSS) in the presence (middle) or absence (left) of detergent (0.05% NP40 and 1% Triton X-100) or a high speed supernatant (HSS) generated in the absence of detergent (right). Equivalent amounts of starting material (input) were used for each immunoprecipitation, and the presence of VPS-20 was detected using VPS-20 specific antibodies by immunoblot analysis. (F–H) ESCRT-II alone (panel D), VPS-20 alone (panel E), or a mixture of ESCRT-II and VPS-20 (F; at a 1:2 molar ratio) were separated by size exclusion chromatography. Stokes radii were calculated based on the elution profile of characterized standards. Peak fractions are highlighted (boxes with dashed lines). (I) A graphical representation of the densitometry measurements conducted on each gel shown in panels D, E, and F. Based on this graph, peak elution volumes were determined to be the following: 12.9 mL for ESCRT-II alone, 15.1 mL for VPS-20 alone, 12.1 mL for ESCRT-II within the mixture of it and VPS-20, and two peaks of 12.2 mL (associated with ESCRT-II) and 14 mL (free of ESCRT-II) for VPS-20 within mixture. (J) A C. elegans extract was separated by size exclusion chromatography, and fractions eluted were immunoblotted using antibodies directed against ESCRT-II (top) and VPS-20 (bottom). The peak fraction in which ESCRT-II and VPS-20 are both found is highlighted (boxes with dashed lines).