Figure 4.

Abrogated binding of Munc18-2 to syntaxin-11 N-terminal R4A or Habc-domain L58P mutations. (A) Dendrogram comparing human syntaxins based on peptide sequence alignments of the N-termini (N-peptide and Habc domain; residues 1-167 of Stx11). (B) Amino acid alignments of human syntaxin (B) N-terminus and (C) Habc domain sequences. Red shade indicates a high degree of sequence homology, whereas blue shade indicates low homology. (D) HEK-293T cells were transfected with empty vector (EV), or constructs encoding FLAG-Stx11 wild-type (wt), R4A, L58P, Q268X, or HA-Munc18-2 wild-type. Twenty-four hours after transfection, WCL were prepared and analyzed for expression of Stx11, Munc18-2, and actin. (E) HEK-293T cells constructs as indicated. Twenty-four hours after transfection, WCL were prepared from Stx11 transfectants and Stx11 was immunoprecipitated to beads. The beads were washed extensively and thereafter incubated with WCL from EV or HA-Munc18-2 wt transfected HEK-293T cells. Beads were washed, proteins eluted, and analyzed by Western blotting with antibodies as indicated.