Figure 5.
Figure 5. How BubR1 acetylation is involved in KT–MT interaction. (A) The HeLa-FRT-BubR1, -K250R, or -K250Q cells were depleted of endogenous BubR1, and the expression of the indicated FLAG-tagged BubR1 constructs was induced by doxycycline treatment. The cells were synchronized in mitosis using the thymidine block and treated with nocodazole/MG132. BubR1 complexes were immunopurified using an anti-FLAG (M2) antibody, followed by WB using the indicated antibodies. The total lysates were included in the WB as an input control. Immunoprecipitated CENP-E bands were normalized to BubR1, and the binding affinities of each BubR1 to CENP-E were determined. The relative binding to CENP-E and the SDs in the graph are from four independent experiments (right). (B) Immunostaining of CENP-E and CREST in WT, K243R/+, and BubR1+/− MEFs after the cold MT assay. The cells were treated with MG132 before fixation. Enlarged images are shown at the bottom. Red, BubR1; yellow, CENP-E; green, α-tubulin. Bars: (yellow) 5 µm; (white) 1 µm. (C) Relative levels of BubR1 and CENP-E from B, in the presence of MG132, as measured by fluorescence intensities at the cellular level (left) and at KTs (right) are represented by bar graphs. Fifty cells in each genotype were scored in two independent experiments (mean ± SEM; n > 500 KTs). (D) WB analysis of CENP-E and BubR1 from lysates prepared from untreated, Noc-treated, or Noc + MG132-treated MEFs. The blot was reprobed with anti-actin antibody as a loading control. (E) GFP-tagged CENP-E expression construct (Kim et al., 2010) or K250Q expression construct was transfected into K243R/+ MEFs. 1 d after transfection, MEFs were synchronized via serum starvation and released into mitosis with change with fresh media supplemented with 16% FBS. 24 h later, cells were treated with MG132 for 2 h and subjected to cold MT assay. Representative images are shown. Ectopic expression of CENP-E-GFP or K250Q-EGFP was detected by green fluorescence. EGFP-expressing construct was transfected for control. Enlarged images of the insets (excluding GFP) are shown at the right. Red, α-tubulin; blue, CREST staining; green, GFP. Bars: (white) 1 µm; (yellow) 5 µm. (F) Comparison of phosphorylated Hec1 in metaphase cells. MEFs from WT, K243R/+, and BubR1+/− mice were treated with 10 µM MG132 for 2 h and subjected to cold MT assay. The levels of pHec1 scored in two independent experiments are shown in bar graphs (mean ± SEM; n > 450 KTs; number of cells scored: WT, n = 23; K243R/+, n = 34; BubR1+/−, n = 25). Representative images are shown at the right with enlarged images of individual KTs in the insets. Bars: (white) 1 µm; (yellow) 5 µm. (G) MEFs were treated with Monastrol for 3 h, washed extensively, and released 1 h in the presence of MG132 and subjected to cold MT assay. Congressed and uncongressed chromosomes were scored in two independent experiments and presented in the bar graphs (mean ± SEM; number of cells scored: WT, n = 71; K243R/+, n = 78; BubR1+/−, n = 73). Asterisks mark significant p-values when compared with WT (*, P = 0.1; **, P = 0.5). (H) Myc-tagged WT-BubR1, K250R, or K250Q expression constructs were transfected into PP2A-B56α–expressing HeLa-FRT cells (Kruse et al., 2013). Expression of PP2A-B56α was induced with doxycycline followed by cell synchronization and mitotic arrest with the treatment of Noc and MG132 to inhibit proteolysis. The cells were subjected to IP and WB. WB of the catalytic subunit of PP2A (PP2A/C) was included for normalization of IP (top). Relative binding affinity to PP2A-B56α was calculated by measuring 9E10 band intensities (WT, K250R, and K250Q, respectively) in FLAG immune complex (PP2A-B56α). SDs in bar graphs are from four independent experiments (bottom). eV, empty vector. (I) EGFP-PP2A-B56α–expressing construct was transfected into K243R/+ MEFs. 24 h later, MEFs were treated with 200 ng/ml Noc for 4 h, and then subjected to chromosome spread and immunofluorescence assay. BubR1 was detected by immunofluorescence and PP2A-B56α by the fluorescence of GFP (left). This experiment was done in the absence of MG132. Fluorescence intensity of PP2A-B56α relative to BubR1 at KT was scored in WT and K243R/+ cells and depicted as a histogram (right). The results are from two independent experiments of >600 KTs (mean ± SEM; number of cells: WT, n = 33; K243R/+, n = 32; P = 0.061). Red, BubR1; green, PP2A-B56α; blue, DAPI. Bars: (white) 1 µm; (yellow) 5 µm.

How BubR1 acetylation is involved in KT–MT interaction. (A) The HeLa-FRT-BubR1, -K250R, or -K250Q cells were depleted of endogenous BubR1, and the expression of the indicated FLAG-tagged BubR1 constructs was induced by doxycycline treatment. The cells were synchronized in mitosis using the thymidine block and treated with nocodazole/MG132. BubR1 complexes were immunopurified using an anti-FLAG (M2) antibody, followed by WB using the indicated antibodies. The total lysates were included in the WB as an input control. Immunoprecipitated CENP-E bands were normalized to BubR1, and the binding affinities of each BubR1 to CENP-E were determined. The relative binding to CENP-E and the SDs in the graph are from four independent experiments (right). (B) Immunostaining of CENP-E and CREST in WT, K243R/+, and BubR1+/− MEFs after the cold MT assay. The cells were treated with MG132 before fixation. Enlarged images are shown at the bottom. Red, BubR1; yellow, CENP-E; green, α-tubulin. Bars: (yellow) 5 µm; (white) 1 µm. (C) Relative levels of BubR1 and CENP-E from B, in the presence of MG132, as measured by fluorescence intensities at the cellular level (left) and at KTs (right) are represented by bar graphs. Fifty cells in each genotype were scored in two independent experiments (mean ± SEM; n > 500 KTs). (D) WB analysis of CENP-E and BubR1 from lysates prepared from untreated, Noc-treated, or Noc + MG132-treated MEFs. The blot was reprobed with anti-actin antibody as a loading control. (E) GFP-tagged CENP-E expression construct (Kim et al., 2010) or K250Q expression construct was transfected into K243R/+ MEFs. 1 d after transfection, MEFs were synchronized via serum starvation and released into mitosis with change with fresh media supplemented with 16% FBS. 24 h later, cells were treated with MG132 for 2 h and subjected to cold MT assay. Representative images are shown. Ectopic expression of CENP-E-GFP or K250Q-EGFP was detected by green fluorescence. EGFP-expressing construct was transfected for control. Enlarged images of the insets (excluding GFP) are shown at the right. Red, α-tubulin; blue, CREST staining; green, GFP. Bars: (white) 1 µm; (yellow) 5 µm. (F) Comparison of phosphorylated Hec1 in metaphase cells. MEFs from WT, K243R/+, and BubR1+/− mice were treated with 10 µM MG132 for 2 h and subjected to cold MT assay. The levels of pHec1 scored in two independent experiments are shown in bar graphs (mean ± SEM; n > 450 KTs; number of cells scored: WT, n = 23; K243R/+, n = 34; BubR1+/−, n = 25). Representative images are shown at the right with enlarged images of individual KTs in the insets. Bars: (white) 1 µm; (yellow) 5 µm. (G) MEFs were treated with Monastrol for 3 h, washed extensively, and released 1 h in the presence of MG132 and subjected to cold MT assay. Congressed and uncongressed chromosomes were scored in two independent experiments and presented in the bar graphs (mean ± SEM; number of cells scored: WT, n = 71; K243R/+, n = 78; BubR1+/−, n = 73). Asterisks mark significant p-values when compared with WT (*, P = 0.1; **, P = 0.5). (H) Myc-tagged WT-BubR1, K250R, or K250Q expression constructs were transfected into PP2A-B56α–expressing HeLa-FRT cells (Kruse et al., 2013). Expression of PP2A-B56α was induced with doxycycline followed by cell synchronization and mitotic arrest with the treatment of Noc and MG132 to inhibit proteolysis. The cells were subjected to IP and WB. WB of the catalytic subunit of PP2A (PP2A/C) was included for normalization of IP (top). Relative binding affinity to PP2A-B56α was calculated by measuring 9E10 band intensities (WT, K250R, and K250Q, respectively) in FLAG immune complex (PP2A-B56α). SDs in bar graphs are from four independent experiments (bottom). eV, empty vector. (I) EGFP-PP2A-B56α–expressing construct was transfected into K243R/+ MEFs. 24 h later, MEFs were treated with 200 ng/ml Noc for 4 h, and then subjected to chromosome spread and immunofluorescence assay. BubR1 was detected by immunofluorescence and PP2A-B56α by the fluorescence of GFP (left). This experiment was done in the absence of MG132. Fluorescence intensity of PP2A-B56α relative to BubR1 at KT was scored in WT and K243R/+ cells and depicted as a histogram (right). The results are from two independent experiments of >600 KTs (mean ± SEM; number of cells: WT, n = 33; K243R/+, n = 32; P = 0.061). Red, BubR1; green, PP2A-B56α; blue, DAPI. Bars: (white) 1 µm; (yellow) 5 µm.

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