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. 2014 Jun 9;9(6):e98900. doi: 10.1371/journal.pone.0098900

Figure 2. Analysis of the effect of FHL-4 mutations on the interaction of syntaxin 11 with SNAP23 and Munc18-2.

Figure 2

(A) Co-immunopreciptation of syntaxin 11 and SNAP23. Prior to lysis, YTS NK cells were pre-incubated in the absence or presence of NEM. Cell lysates were then incubated in the presence or absence of a mouse syntaxin 11 specific antibody and antibody bound proteins pulled down with protein-G sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and rabbit syntaxin 11 specific antibodies. (B) GST pulldowns with syntaxin 11 FHL-4 mutants. GST, GST-syntaxin 11 and GST fusions of FHL-4 mutants were bound to glutathione sepharose beads (See Figure S1 for a coomassie stained gel of the GST fusions bound to the glutathione sepharose beads). Pulldowns of a cell lysate prepared from HeLa-M cells transfected with Myc-Munc18-2 were then performed with GST and the GST-fusions immobilized on glutathione sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and Myc-tag specific antibodies.