JCS027417 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Localization of UBPY, AMSH and ubiquitylated proteins in COS-7 and NIH-3T3 cells during cytokinesis. COS-7 (A-A′′,C-C′′,E-E′′) and NIH-3T3 (B-B′′,D-D′′,F-F′′) cells at stage 2 (C-F′′) or 4 (A-B′′) of cytokinesis were stained with anti-UBPY (A,B), anti-AMSH (C,D) or FK2 (E,F) antibody, together with anti-tubulin antibody (A′-F′). A′′-F′′ are merged images. Insets show high-magnification images of the central spindle/midbody regions. Scale bars: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Competition of FK2 staining at the central spindle by an excess of Ub chains. HeLa cells at stage 2 of cytokinesis were stained with FK2 which was pre-incubated without (A) or with (B,C) an excess of Lys⁴⁸-linked (B) and Lys⁶³-linked (C) Ub chains. A′-C′ are merged images with anti-tubulin (red) and TO-PRO-3 (blue) staining. Insets show high-magnification images of the central spindle region. Scale bars: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Localization of Hrs and endosome markers during cytokinesis. (A-A′′) HeLa cells at stage 2 of cytokinesis were double-stained with anti-Hrs (A) and anti-tubulin (A′) antibodies. A′′ is a merged image. (B-D) HeLa cells at stage 4 of cytokinesis were stained with anti-EEA1 (B, green), anti-LAMP1 (C, green) or anti-LBPA (D, green) antibody, together with anti-tubulin antibody (red) and TO-PRO-3 (blue). Cytoplasmic punctate staining in green indicates endosomes. Insets show high-magnification images of the central spindle/midbody regions. Scale bars: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Ubiquitylation/deubiquitylation and SNARE activity of VAMP8. (A) HeLa cells were transfected with FLAG-tagged UBPY, UBPY^C748A, AMSH or AMSH^D348A. Endogenous VAMP8 was immunoprecipitated from their lysates with anti-VAMP8 antibody, and immunoblotted with FK2 (top) and anti-VAMP8 (middle) antibodies. Expression levels of the FLAG-tagged DUB constructs were assessed by immunoblotting of the total lysates with anti-FLAG antibody (bottom). The band 3 in the top panel indicates endogenous VAMP8 conjugated with 3 Ub molecules, which migrated slightly faster than FLAG-tagged VAMP8 (Figs 5 and 6) owing to the lack of the FLAG epitope. Similar to FLAG-VAMP8, endogenous VAMP8 migrated as a doublet band. We were not able to determine whether VAMP8 is conjugated with two Ub molecules because it co-migrated with the IgG light chain (IgG-L) used for immunoprecipitation. Mono-ubiquitylated VAMP8 was undetectable. (B) HeLa cells were transfected with or without HA-VAMP8 together with FLAG-tagged syntaxin 2 or SNAP-23. Syntaxin 2 and SNAP-23 were immunoprecipitated from their lysates with anti-FLAG antibody, and immunoblotted with FK2 (top), anti-HA (second from top) and anti-FLAG (third from top) antibodies. The expression level of HA-VAMP8 was assessed by immunoblotting of the total lysates with anti-HA antibody (bottom). The bands 2 and 3 in the top panel indicate VAMP8 proteins conjugated with two or three Ub molecules.