Fig. 5. VAMP8 undergoes ubiquitylation. (A-B'') HeLa cells were transfected without (A-A'') or with (B-B'') FLAG-VAMP8, and stained with anti-VAMP8 (A) or anti-FLAG (B) antibody, together with FK2 (A',B', red) and TO-PRO-3 (A', blue), at stage 2 or 3 of cytokinesis. A'' and B'' are merged images. Insets show high magnification images of the central spindle region. Scale bar: 10 µm. (C) Schematic structure of mouse VAMP8 and its mutants used in this study. Positions of the SNARE motif, transmembrane domain and the seven Lys residues are indicated. (D) FLAG-tagged VAMP8 and its mutants were transfected into HeLa cells, and immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were immunoblotted with anti-FLAG antibody (left) or FK2 (right). Black arrowheads indicate the positions of non-ubiquitylated FLAG-VAMP8 (band 0, 14 kDa) and FLAG-VAMP8 proteins conjugated with 1 (band 1, 22 kDa), 2 (band 2, 30 kDa) and 3 (band 3, 38 kDa) Ub molecules. Asterisks indicate the IgG light (L) chain used for immunoprecipitation. The identity of the bands indicated by a white arrowhead is unknown. Ubiquitylated VAMP8 was often detected as a doublet band. The reason for this is unknown.