Supplementary Figures 1 - 6 from Deubiquitination of γ-Tubulin by BAP1 Prevents Chromosome Instability in Breast Cancer Cells

Supplementary Figure 1. The cellular distribution of gamma-tubulin and BRCA1 in MCF10A cells, using immunofluorescence staining and confocal microscopy. Bars, 10 mm. Supplementary Figure 2. Confidence view screenshot from STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) shows known and predicted protein-protein interaction network of human BAP1 and UCHL1, as well as gamma tubulin (TUBG1) and BRCA1, which were used as inputs to the query. Supplementary Figure 3. (A)The subcellular distribution of endogenous BAP1 in MCF10A cells, using immunofluorescence staining and confocal microscopy.(B) The subcellular localization of BAP1 in MCF10A cells were biochemically divided into cytosolic (Cyto), Nuclear membrane (NM) and Nucleus (Nu) fractions and examined by Western blotting with an anti-BAP1 antibody. alpha-tubulin and Lamin B were used as molecular markers for the cytosolic and Nucleus fractions, respectively. Supplementary Figure 4. (A) Immunoprecipitation of endogenous alpha-tubulin from synchronized MCF10A cells and immunoblotting using UCHL1 and alpha-tubulin antibodies. The arrowhead points to a shift in size of alpha-tubulin corresponding to ubiquitinated alpha-tubulin.MCF10A cells were synchronized by double-thymidine block release and collected in G1, S and G2/M phases. (B) MCF10A cells were synchronized by a double thymidine block. Cells in mitotic phase were obtained by nocodazole treatment (M0) and released from nocodazole after 45 min (M1), 90 min (M2), and 180 min (M3). Cell extracts were immunoprecipitated with alpha-tubulin antibody and analyzed by Western blotting using antibodies against UCHL1, alpha-tubulin, and actin. The lysate (lower panel) shows an equal amount of protein used for immunoprecipitation. Supplementary Figure 5. (A) Western blot analysis of BAP1 and Actin expression in CAMA and T47D cells. (B) Immunoprecipitation of endogenous BAP1 from synchronized CAMA and T47D cells and immunoblotting using anti-BAP1 and alpha-tubulin antibodies. Cells were synchronized by double-thymidine block release and collected in mitosis by mitotic shake-off. Both asynchronous and synchronous cell extracts were used for immunoprecipitation. The lysate (lower panel) shows an equal amount of protein used for immunoprecipitation (C) Analysis of the levels of BAP1 and ubiquitinated alpha-tubulin in CAMA and T47D cells treated for three days with shRNA against BAP1 or empty expression plasmid (shCtrl). The arrowhead points to a shift in size of alpha-tubulin corresponding to ubiquitinated alpha-tubulin. Supplementary Figure 6. (A) Western blot analysis of wild-type, Flag-HA-BAP1, using HA antibody. (B) Western blot analysis of, Flag-BAP1-C91A, using flag antibody.