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. Author manuscript; available in PMC: 2015 May 4.
Published in final edited form as: Biochem J. 2014 Dec 1;464(2):251–258. doi: 10.1042/BJ20140845

Figure 5. Munc18-1 and Munc18c bind non-competitively to the Doc2b scaffold.

Figure 5

(A) Schematic representation of the GST-Doc2b competition assay. GST-Doc2b-linked beads were pre-incubated with one of the two Munc18 isoforms for 2h at 4°C. Binary complexes were pelleted to eliminate unbound protein for subsequent addition of the other Munc18 isoform. (B) GST-Doc2b was preincubated with Munc18-1 followed by addition of Munc18c in excess of 1 (+), 2 (++) or 3 (+++) molar equivalents to the binding reaction. (C) GST-Doc2b was preincubated with Munc18c followed by addition of Munc18-1, as described in panel (B). Both bound and unbound proteins were captured and resolved by 10% SDS-PAGE for immunoblot detection (IB). Data are representative of at least four independent experiments using three or more independent batches of recombinant proteins.