Figure 1.
VPS33B binds to the SNARE domain of endosomal Stx12. (A) GST-pulldown assay. GST fusion proteins corresponding to the whole cytosolic domain of each of the 13 human Syntaxins (Qa-SNAREs) were bound to glutathione beads and incubated with purified Twin Strep-VPS16B/His-VPS33B complex. Top panel, the bound Twin Strep-VPS16B/His-VPS33B complex was detected by immunoblotting (IB) with antibodies against the His-VPS33B polyhistidine tag (His). Bottom panel, SDS-PAGE (Coomassie), indicating comparable loading amounts of the GST-fusion proteins. (B) Coimmunoprecipitation assay. A total MEG-01 cell extract was incubated with rabbit anti-Stx12 antibody bound to Sepharose beads or irrelevant rabbit IgG bound to Sepharose beads as a control. Bound proteins were analyzed by IB with antibodies against VPS33B and Stx12. (C) Cartoon depicting Stx12 domains. (D) GST-pulldown assay with the whole Stx12 cytosolic domain (Cyt) or the indicated fragments (N-Pep, Habc, SNARE) fused to GST and purified Twin Strep-VPS16B/His-VPS33B complex. The bound Twin Strep-VPS16B/His-VPS33B complex was detected by IB with antibodies to both VPS33B and VPS16B. SDS-PAGE (Coomassie) indicates comparable loading amounts of the GST-fusion proteins. (E) Quantification of the GST pulldown shown in D. The bars indicate the ratio of VPS33B pulled down by each of the Stx12 fragments over the whole cytosolic domain of Stx12 (n = 3). All samples were compared with GST alone, and statistical significance was determined via unpaired, 2-tailed Student t test. (F) GST-pulldown assay with the SNARE domains of Stx4, Stx5, and Stx12 fused to GST and purified Twin Strep-VPS16B/His-VPS33B complex. The bound Twin Strep-VPS16B/His-VPS33B complex was detected by IB with an antibody against VPS33B. SDS-PAGE (Coomassie) indicates comparable loading amounts of the GST-fusion proteins. (G) Quantification of the GST pulldown shown in F. The bars indicate the ratio of VPS33B pulled down by each of the SNARE fragments over GST (n = 3). All samples were compared with GST alone, and statistical significance was determined via unpaired, 2-tailed Student t test.

VPS33B binds to the SNARE domain of endosomal Stx12. (A) GST-pulldown assay. GST fusion proteins corresponding to the whole cytosolic domain of each of the 13 human Syntaxins (Qa-SNAREs) were bound to glutathione beads and incubated with purified Twin Strep-VPS16B/His-VPS33B complex. Top panel, the bound Twin Strep-VPS16B/His-VPS33B complex was detected by immunoblotting (IB) with antibodies against the His-VPS33B polyhistidine tag (His). Bottom panel, SDS-PAGE (Coomassie), indicating comparable loading amounts of the GST-fusion proteins. (B) Coimmunoprecipitation assay. A total MEG-01 cell extract was incubated with rabbit anti-Stx12 antibody bound to Sepharose beads or irrelevant rabbit IgG bound to Sepharose beads as a control. Bound proteins were analyzed by IB with antibodies against VPS33B and Stx12. (C) Cartoon depicting Stx12 domains. (D) GST-pulldown assay with the whole Stx12 cytosolic domain (Cyt) or the indicated fragments (N-Pep, Habc, SNARE) fused to GST and purified Twin Strep-VPS16B/His-VPS33B complex. The bound Twin Strep-VPS16B/His-VPS33B complex was detected by IB with antibodies to both VPS33B and VPS16B. SDS-PAGE (Coomassie) indicates comparable loading amounts of the GST-fusion proteins. (E) Quantification of the GST pulldown shown in D. The bars indicate the ratio of VPS33B pulled down by each of the Stx12 fragments over the whole cytosolic domain of Stx12 (n = 3). All samples were compared with GST alone, and statistical significance was determined via unpaired, 2-tailed Student t test. (F) GST-pulldown assay with the SNARE domains of Stx4, Stx5, and Stx12 fused to GST and purified Twin Strep-VPS16B/His-VPS33B complex. The bound Twin Strep-VPS16B/His-VPS33B complex was detected by IB with an antibody against VPS33B. SDS-PAGE (Coomassie) indicates comparable loading amounts of the GST-fusion proteins. (G) Quantification of the GST pulldown shown in F. The bars indicate the ratio of VPS33B pulled down by each of the SNARE fragments over GST (n = 3). All samples were compared with GST alone, and statistical significance was determined via unpaired, 2-tailed Student t test.

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