EMBO/soda-nel huggingface dataset:
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 5

Figure 5. Inhibition of the Igf pathway mimics Igfbp1a’s stimulatory effect on β-cell regeneration.

(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group. (I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a. (L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 & EV3.
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 6

Figure 6. Igfbp1a also increases β-cell regeneration in 1-month-old zebrafish.

(A-B) Representative confocal projections of the whole pancreas (dashed lines) of 35-day-old Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), that were subjected to β-cell ablation between day 30-31 and then allowed to regenerate for 4 days. Scale bars represent 100 μm. (C) Body length of control and Tg(bactin:igfbp1a) zebrafish; P<0.0001. (D-H) Quantification of β-cell regeneration was automated with an ImageJ script. (D) Insulin-positive area per zebrafish. (E) Relative insulin-positive area per body length; P<0.01. (F) The percentage of the pancreas area that was insulin-positive was significantly larger in Tg(bactin:igfbp1a) than in controls; P<0.0001. (G) The total number of recorded units of adjacent ins:H2B-GFP+ pixels (units ranging from single β cells to β-cell clusters) did not differ between control and Tg(bactin:igfbp1a) zebrafish. (H) The size of recorded ins:H2B-GFP+ units was on average larger in Tg(bactin:igfbp1a) than in controls; P=0.039. n=26 in the control group, n=27 in the Tg(bactin:igfbp1a) group.
IGFBP1 Increases β-cell Regeneration by Promoting α- to β-cell Transdifferentiation (2016)
Figure 7

Figure 7. IGFBP1 increases the prevalence of insulin- and glucagon-expressing bihormonal cells in both mouse and human islets.

Isolated islets from mice and from human donors were treated for 3 days with recombinant mouse or human IGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa. (H) Our proposed mechanism by which the extracellular signals insulin, IGFs and IGFBP1 can regulate α- to β-cell transdifferentiation.
FLIP-mediated autophagy regulation in cell death control (2009)
figf1

(a–c) At 12–16 h post-transfection with GFP–LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2–4 h (a) or with 2 μM rapamycin for 1–3 h (b, c). Subsequently, GFP–LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP–LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200–300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP–LC3 (c). (d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c. (e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2–4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody. (g) At 12–16 h post-transfection with GFP–LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP–LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP–LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200–300 cells; three independent experiments; *P 0.01). (h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.
FLIP-mediated autophagy regulation in cell death control (2009)
figf2

(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel). (b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody. (c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST–Atg3 along with Flag–vFLIP (left) or GST or GST–Atg3 along with GFP–LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies. (d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST–vFLIP DED1 mutants (middle) or GST–vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody. (e) At 48 h post-transfection with GST, GST–Atg3, and either Flag–vFLIP or Flag–vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody. (f) At 48 h post-transfection with GST–Atg3 and GFP–LC3 along with increasing amount of Flag–vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody. (g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a–g).
FLIP-mediated autophagy regulation in cell death control (2009)
figf3

(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death. (b) At 12–16 h post-transfection with GFP–LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP–LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200–300 cells; three independent experiments; *P 0.01. (c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200–300; three independent experiments; *P 0.05. (d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001. (e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b–e).
FLIP-mediated autophagy regulation in cell death control (2009)
figf4

(a) Sequence alignment of the FLIP α2 and α4 peptide sequences. The red letters correspond to hydrophobic core residues. (b) At 12–16 h post-transfection with GFP–LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05. (c) The details are identical to those described in b and GFP–LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm. (d) KSHV-infected BCBL1 cells were treated with 30 μM of the K-α2 or the K-α4 peptide for 12 h and subjected to scanning electron microscopy. Scale bar, 50 nm. (e) TREX-BCBL-Vector and TREX-BCBL-vFLIP cells were treated with the K-α and the K-α4 (30 μM each) peptide for 12 h, followed by immunoblotting (IB) with anti-LC3 and anti-actin antibody. (f) NOD/SCID mice received an injection of 5 × 106 BCBL1–Luc cells, followed by intraperitoneal injections with 300 μg the TAT, K-α2 or Kα-4 peptide for three weeks (top three panels). After three weeks, a group of three mice treated with the TAT peptide was challenged with 300 μg of the K-α2 or Kα-4 peptide for an additional three weeks (bottom panel, see Supplementary Information, Fig. S15). Tumours were measured by in vivo bioluminescence imaging.
FLIP-mediated autophagy regulation in cell death control (2009)
figf5

(a) At 16 h post-transfection with GST, GST-K-α2 or GST-K-α4 along with Flag–vFLIP (top) or Flag–cFLIPL (bottom), HEK293T cells were used for GST pulldown, followed by immunoblotting (IB) with anti-Flag or anti-FLIP antibody. Whole-cell lysates (WCLs) were used for IB with anti-GST, anti-Flag or anti-FLIP antibody. (b) At 16 h post-transfection with V5-Atg3 and Flag–vFLIP with increasing amounts of GST–K-α2 or GST–K-α4, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-V5 antibody or for immunoprecipitation (IP) with anti-V5 antibody, followed by IB with anti-Flag antibody. WCLs were used for IB with anti-GST, anti-Flag or anti-V5 antibody. (c) At 12–16 h post-transfection with GST–Atg3 along with Flag–vFLIP (first), Flag–cFLIPS (second), or HA–LC3 (third) with increasing amounts of the K-α2+K-α4 peptides (top three panels) or C-α2+C-α4 peptides (bottom panel), HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-HA antibodies. WCLs were used for IB with anti-GST and anti-Flag antibody. (d) At 12–16 h post-transfection with Flag–vFLIP, HA–LC3 and V5-Atg3, HEK293T cells were treated with increasing amounts of the K-α2+K-α4 peptides and used for IP anti-V5 antibody, followed by IB with anti-Flag or anti-HA antibody. WCLs were used for IB with anti-V5, anti-HA and anti-Flag antibody. (e) Model of FLIP- and its peptide-mediated deregulation of autophagy.
Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint (2022)
Figure 1

Figure 1. Cyclin B2 localizes at unaligned kinetochores in HeLa cells.

(A) Schematic representation of human Cyclin B1 and Cyclin B2 protein domain organization.

(B) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.

(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.

(D and E) Representative immunofluorescence images of HeLa cells treated with an indicated siRNA (D) or various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.

(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.
Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint (2022)
Figure 2

Figure 2. Mad2 recruits Cyclin B2 to kinetochores.

(A) Table showing the proteins binding to Cyclin B1 and Cyclin B2 identified by immunoprecipitation and subsequent mass spectrometry. In brief, Flag-Cyclin B1 and Flag-Cyclin B2 were expressed in 293T cells and immunoprecipitated with anti-Flag M2 beads. Then Cyclin B1/B2-bound beads were incubated with pre-cleared mitotic HeLa cell lysates. The immunoprecipitates were analyzed by mass spectrometry.

(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747).

(C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody.

(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining.

(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments). Student's t-test was used to calculate P-values. ****P < 0.0001; n.s., not significant.
Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint (2022)
Figure 3

Figure 3. Cyclin B2 C-terminal MIM is required for its kinetochore localization.

(A) Multiple sequence alignment of the Cyclin B2 and Cyclin B1 proteins from various species as indicated. The sequence alignment was developed by ClustalW2 software.

(B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody.

(C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody.

(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). Scale bar, 10 µm.

(E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments). Student's t-test was used to calculate P-values. ***P < 0.001.
Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint (2022)
Figure 4

Figure 4. Cyclin B2 kinetochore localization is required for faithful mitotic progression.

(A) HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were collected and lysed. The samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B2 antibody, anti-Cyclin B1 antibody, and anti-tubulin antibody.

(B) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA. Images were acquired at the indicated time points after the start of NEBD. The arrow indicates lagging chromosomes. Scale bar, 10 µm.

(C) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B. Bars are means ±SD (≥30 cells from three independent experiments). Student's t-test was used to calculate P-values. ***P < 0.001.

(D) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B (n = 30 cells). Bars indicate means ± SD. Student's t-test was used to calculate P-values for comparison of siControl and siCyclin B2 cells; n.s., not significant.

(E) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. Images were acquired at the indicated time points after the start of NEBD. Arrows indicate a chromosome bridge. Scale bar, 10 µm.

(F) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E. Bars are means ±SD (≥30 cells from three independent experiments). Student's t-test was used to calculate P-values. ***P < 0.001.
Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint (2022)
Figure 5

Figure 5. Cyclin B2 is required for robust SAC signalling.

(A-F) (A and D) Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in A, Cdc20 (green) in D, ACA (red), and DNA (blue). Scale bar, 10 µm. (B and E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in B, Cdc20 (red) in E, and DNA (blue). Scale bar, 10 µm. (C and F) Scatter graphs illustrating kinetochore intensity of Mad2 (C, cells treated as in A and B) and Cdc20 (F, cells treated as in D and E). Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments). Student's t-test was used to calculate P-values. ****P < 0.0001.

(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h. Values are means ± SD (≥1000 cells from three independent experiments). Student's t-test was used to calculate P-values. ****P < 0.0001.

(H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies.