T H E J O U R N A L O F C E L L B I O L O G Y

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Cornelia Washington
6 years ago
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T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Krenn et al., http://www.jcb.org/cgi/content/full/jcb.201110013/dc1 Figure S1.

1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Krenn et al., Figure S1. Levels of expressed proteins and demonstration that C-terminal and N-terminal EGFP fusions behave indistinguishably. (A) Western blots of proteins extracts from HeLa cells transfected with Bub1-contraining plasmids shown in Fig. 1 B. (B) Same as in A for additional images shown in Fig. 1 B. Note that the Bub1 antibody recognizes all N-terminally deleted versions of Bub1 but not Bub Vinculin or Bub3 were used as a loading control. (A and B) Single asterisks represent degradation bands detected by the mouse anti-gfp antibody used. (C) Western blot of protein extracts from HeLa cells transfected with BubR1-containing plasmids shown in Fig. 1 C. Single and double asterisks represent degradation bands and unspecific bands, respectively, detected by the rabbit anti-gfp antibody. (D) Same as in A for additional images shown in Fig. 1 C. Bub3 was used as a loading control. (E) Immunofluorescence images of mitotic HeLa cells expressing C-terminally EGFP-fused Bub1 (Bub1 189-EGFP). Cells were stained with DAPI (DNA) and CREST sera (kinetochores). The inset shows a higher magnification of kinetochore regions (box). Bar, 5 µm. (F) Western blot of HeLa cells used in E. Vinculin was used as a loading control. end, endogenous; MM, molecular mass. S1

2 Figure S2. Comparison of the KI1 Bub1 and KI2 BubR1 structures. (A) Cartoon representation of the BubR1 KI2 complex (Protein Data Bank no. 3SI5) with highlights of important residues at the complex interface. (B) As in A, with the rotated complex. (C) Cartoon representation of the Bub1 KI complex (this study) with highlights of important residues at the complex interface. (D) As in C, with the rotated complex. S2

Figure S3. Fluorescence images of mitotic cells expressing wild-type Bub1 or BubR1 and single point mutants and localization experiments in the absence of endogenous Bub1.

3 Figure S3. Fluorescence images of mitotic cells expressing wild-type Bub1 or BubR1 and single point mutants and localization experiments in the absence of endogenous Bub1. (A and B) HeLa cells were transiently transfected with plasmids containing N-terminally EGFP-tagged wild-type (WT) Bub1 (A) and BubR1 (B) or their mutants. (C) Schematic description of the protocol used in D and E. In brief, cells were transfected with plasmids carrying EGFP alone or EGFP-tagged Bub1 constructs. All Bub1 constructs were mutated to be resistant to RNAi-based depletion. Cells were then depleted of endogenous Bub1 by RNAi (see Materials and methods for details) followed by double thymidine arrest (DTA). 5 h after release from the arrest, cells were treated with nocodazole and then processed for Western blotting (WB) or immunofluorescence (IF). (D) Western blot of extracts from cells treated as in C. Bub3 was used as a loading control. Note that anti-bub1 antibody recognized both endogenous and RNAi-resistant EGFP-tagged Bub1 proteins. (E) Immunofluorescence images of mitotic cells treated as described in C. Cells were stained with DAPI (DNA) and CREST sera (kinetochores). Insets show a higher magnification of kinetochore regions (boxes). MM, molecular mass; IB, immunoblot. Bars, 5 µm. S3

4 Figure S4. Additional kinase assays. (A and B) In vitro kinase activity toward histone H2A of recombinant Bub1 Bub3 (A) or EGFP and EGFP-fused Bub1 immunopurified from Flp-In T-REx cells (B) in the presence of 2OH-BNPP1 inhibitor. (C) In vitro kinase activity toward histone H2A of recombinant Bub1 Bub3 and kinase-dead mutant carrying the K821R mutation. KD, kinase dead. (D) Western blot showing the levels of the mitotic marker P-S10-H3 in Flp-In T-REx cells expressing EGFP-tagged versions of Bub1 used for the assay in Fig. 7. In the first lane from the left, extracts from cells expressing GFP and treated with 330 nm nocodazole for 16 h were used as a positive control for the phosphorylation of Ser10 of H3. Bub3 was used as a loading control. Note that Bub1 antibody recognizes all Bub1 versions. Cyc, cycling. AR, autoradiography; CB, Coomassie brilliant blue; WT, wild type; end, endogenous; IB, immunoblot; MM, molecular mass; Mit, nocodazole-treated mitotic cells. S4

5 Figure S5. Additional localization experiments. (A) Immunofluorescence images of mitotic HeLa cells expressing different Bub1 constructs. (B and C) Western blot of protein extracts from HeLa cells transfected with plasmids shown in Fig. 8 (B and C), respectively. Single asterisks represent degradation bands detected by the anti-gfp antibody. (D) Images of mitotic HeLa cells expressing EGFP-tagged Bub1( ) wild type and a mutant carrying the E248K substitution. (E) Western blot of protein extracts from HeLa-expressing cells transfected with plasmids used in D. Vinculin was used as a loading control. Cells were stained with DAPI (DNA) and CREST sera (kinetochores). Insets show a higher magnification of kinetochore regions (boxes). MM, molecular mass; FL, full length; WT, wild type. Bars, 5 µm. S5

Nature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1

Nature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1 Supplementary Figure 1 Mutational analysis of the SA2-Scc1 interaction in vitro and in human cells. (a) Autoradiograph (top) and Coomassie stained gel (bottom) of 35 S-labeled Myc-SA2 proteins (input)