Figure 6. VAMP4 interacts with STX7,... | Rockefeller University Press

Figure 6.

$VAMP4 interacts with STX7, STX8, and VTI1B to form a SNARE complex on lysosomes. (A) GST and GST-tagged VAMP4 were purified by Glutathione Sepharose beads, and His-tagged STX7, STX8, and VTI1B were purified by Ni beads from E. coli. The purified proteins were determined by SDS–PAGE and Coomassie blue staining (n = 3 biological independent experiments). (B) Representative Western blot images showing the interaction of VAMP4 with STX7, STX8, and VTI1B determined by GST pulldown assay in vitro. His-tagged STX7, STX8, and VTI1B were immunoblotted (IB) with anti-His antibody, and GST and GST-tagged VAMP4 were immunoblotted with anti-VAMP4 antibody. GST protein was used as a negative control in the GST pulldown assay (n = 3 biological independent experiments). (C) The association of VAMP4, STX7, STX8, and VTI1B was detected by endogenous co-IP and immunoblotting with the indicated antibodies. IgG was used as a negative control in the co-IP assay. 3% of cell lysates and 10% of the immunoprecipitates were loaded (n = 4 biological independent experiments). (D) Representative Western blot images showing the expression levels of VAMP4, STX7, STX8, and VTI1B in the enriched lysosomal fraction. 15 μg of protein was loaded in each lane (n = 3 biological independent experiments). (E) The association of VAMP4, STX7, STX8, and VTI1B on lysosomes was detected by endogenous co-IP using enriched lysosomal fractions and immunoblotting with the indicated antibodies. IgG was used as a negative control in the co-IP assay. 3% of the isolated lysosomal fraction and 10% of the immunoprecipitates were loaded (n = 3 biological independent experiments). (F–H) INS-1 cells were transfected with nYFP-tagged VAMP4, cYFP-tagged STX7, and nYFP-tagged STX7 (F), cYFP-tagged STX8 and nYFP-tagged STX8 (G), cYFP-tagged VTI1B and nYFP-tagged VTI1B (H), and mCherry-tagged LAMP1 and mCherry-tagged (pro)insulin plasmids for 48 h under live-cell imaging. The interaction of nYFP-tagged VAMP4 with cYFP-tagged STX7, cYFP-tagged STX8, or cYFP-tagged VTI1B was detected by BiFC assay. The plasmids of nYFP-tagged STX7, nYFP-tagged STX8, and nYFP-tagged VTI1B were used as a negative control. mCherry-tagged LAMP1 (red) and mCherry-tagged (pro)insulin (red) represent lysosomes and iISGs, respectively. Scale bars, 5 μm (F–H) and 1 μm (insets in F–H). Source data are available for this figure: SourceData F6.$

VAMP4 interacts with STX7, STX8, and VTI1B to form a SNARE complex on lysosomes. (A) GST and GST-tagged VAMP4 were purified by Glutathione Sepharose beads, and His-tagged STX7, STX8, and VTI1B were purified by Ni beads from E. coli. The purified proteins were determined by SDS–PAGE and Coomassie blue staining (n = 3 biological independent experiments). (B) Representative Western blot images showing the interaction of VAMP4 with STX7, STX8, and VTI1B determined by GST pulldown assay in vitro. His-tagged STX7, STX8, and VTI1B were immunoblotted (IB) with anti-His antibody, and GST and GST-tagged VAMP4 were immunoblotted with anti-VAMP4 antibody. GST protein was used as a negative control in the GST pulldown assay (n = 3 biological independent experiments). (C) The association of VAMP4, STX7, STX8, and VTI1B was detected by endogenous co-IP and immunoblotting with the indicated antibodies. IgG was used as a negative control in the co-IP assay. 3% of cell lysates and 10% of the immunoprecipitates were loaded (n = 4 biological independent experiments). (D) Representative Western blot images showing the expression levels of VAMP4, STX7, STX8, and VTI1B in the enriched lysosomal fraction. 15 μg of protein was loaded in each lane (n = 3 biological independent experiments). (E) The association of VAMP4, STX7, STX8, and VTI1B on lysosomes was detected by endogenous co-IP using enriched lysosomal fractions and immunoblotting with the indicated antibodies. IgG was used as a negative control in the co-IP assay. 3% of the isolated lysosomal fraction and 10% of the immunoprecipitates were loaded (n = 3 biological independent experiments). (F–H) INS-1 cells were transfected with nYFP-tagged VAMP4, cYFP-tagged STX7, and nYFP-tagged STX7 (F), cYFP-tagged STX8 and nYFP-tagged STX8 (G), cYFP-tagged VTI1B and nYFP-tagged VTI1B (H), and mCherry-tagged LAMP1 and mCherry-tagged (pro)insulin plasmids for 48 h under live-cell imaging. The interaction of nYFP-tagged VAMP4 with cYFP-tagged STX7, cYFP-tagged STX8, or cYFP-tagged VTI1B was detected by BiFC assay. The plasmids of nYFP-tagged STX7, nYFP-tagged STX8, and nYFP-tagged VTI1B were used as a negative control. mCherry-tagged LAMP1 (red) and mCherry-tagged (pro)insulin (red) represent lysosomes and iISGs, respectively. Scale bars, 5 μm (F–H) and 1 μm (insets in F–H). Source data are available for this figure: SourceData F6.