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

Figure 3. The N-terminal 24 residues of syntaxin 11 are necessary for binding to Munc18-2 and for the stabilisation of syntaxin 11 expression by Munc18-2.

Figure 3

(A) GST pulldowns with the syntaxin 11ΔN24 mutant. GST and GST-syntaxin 11ΔN24 were bound to glutathione sepharose (See Figure S2 for a coomassie blue stained gel of the proteins bound to glutathione sepharose). Pulldowns of a cell lysate prepared from HeLa-M cells transfected with Myc-Munc18-2 were then performed with the GST and GST-syntaxin 11ΔN24 immobilized on glutathione sepharose The precipitated proteins were resolved by SDS-PAGE and analysed by immunoblotting with SNAP23 and Myc-tag specific antibodies. (B) Flow cytometric analysis of the effect of Munc18-2 on the expression of syntaxin 11. Constructs encoding GFP-syntaxin 11 or GFP-syntaxin 11ΔN24 were transfected into HeLa-M cells with either a control plasmid construct or a construct encoding Myc-Munc18-2. The mean fluorescence value for the cells was quantified by flow cytometry. Statistical analysis was performed using the Student's t-test. * P = 0.032. Error bars represent standard error of the mean for triplicate samples from 3 independent experiments. AU (arbitrary units). (C) Immunoblot analysis of the effect of Munc18-2 on the expression of syntaxin 11. Cell lysates prepared from the HeLa-M transfectants were analysed by immunoblotting with syntaxin 11, Myc-tag and GAPDH specific antibodies.