SipA mimics as a cognate R-SNARE of Syn8. (A) Bioinformatics analyses of the protein sequences of SipA, Syn8, Syn7, Syn13, and VAMP8 were performed using NCBI COBALT to determine the presence of SMs in SipA. (B) To determine the region of SipA interacting with Syn8, GST-Syn8 was immobilized, and beads were incubated with His6-SipA1–435, His6-SipA436–685, His6-SipA1–169, His6-SipA1–242, His6-SipA1–277, or His6-SipA48–277. Binding of different SipA-truncated proteins with Syn8 was detected by Western blot analysis using anti-His antibody. (C) SipA1–169 and SipA1–242 were analyzed by nonreducing SDS-PAGE as indicated to determine the formation of homodimers. Arrows indicate the multimers of indicated proteins. (D) To demonstrate that SipA binds with Syn8 through the SM, SipAR204Q and SipA1-435R204Q were generated, and binding of His6-SipAR204Q or His6-SipA1-435R204Q with GST-Syn8 was determined as described in the Bioinformatics analysis section of Materials and methods. . All results are representative of three independent experiments.
