Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: HeLa cells were transiently transfected with a control siRNA or two independent siRNA oligos (Bub1 si5 and si6) against Bub1 and the following experiments were conducted: (A) Total cell lysates were harvested 36 hours after transfection followed by immunoblotting with the indicated antibodies. (B) Cells were treated with 4Gy of IR. At 24 or 48 hours after IR, cells were stained with the MTT-based proliferation assay. Shown are the averages of relative survival rates, and * indicates statistical significance (P=0.001, T-test). (C) Cells were treated with indicated doses of IR and colony formation assays were conducted. Shown are the averages of at least triplicate samples. Standard errors are shown by error bars. (D) Cells were plated onto cover-slips and after IR cells were fixed and stained with the anti-Histone λ-H2AXantibody followed by immunofluorescence microscopy. Nuclear H2AX foci were counted and shown are the averages of 100 cells. Standard errors are shown by error bars. Statistical analyses were conducted using T-test. (E) The Single Cell Gel Electrophoresis assay was conducted and Olive Tail Moment was recorded. Shown are the averages of 100 cells. Standard errors are shown by error bars.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Control, Western Blot, Staining, Proliferation Assay, Immunofluorescence, Microscopy, Single Cell Gel Electrophoresis

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: (A) HeLa cells transfected with control or Bub1 siRNA were treated with mock or IR (4Gy). Total cell lysates were collected followed by immunoblotting using indicated antibodies. (B) HeLa cells stably transfected with control shRNA or ATM shRNA were treated with mock or IR (4Gy). Total cell lysates were collected followed by immunoblotting using indicated antibodies.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Control, Western Blot, Stable Transfection, shRNA

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: Total cell lysates were harvested from: (A) HeLa cells treated with mock or IR (4Gy); (B) Camptothecin (CPT); (C) HeLa cells treated with mock or IR (4Gy) in the presence or absence of KU55933; (D) SV-40 transformed fibroblast cells lines GM0637 and GM9607 treated with mock or IR (4Gy); (E) HeLa cells transiently transfected with either control siRNA or ATM siRNA followed by mock or IR treatment. Western blot analyses were conducted using indicated antibodies. and (F) The in vitro kinase assay using ATM fragments (either GST-tagged N-terminal a.a. 248-522 or C-terminal fragments of ATM a.a. 2709-2964) in the presence of non-phosphorylated form of Bub1 peptides. The blot was stained by coomassie blue and immunoblotted with the anti-phospho-Ser314 Bub1 antibody.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transformation Assay, Transfection, Control, Western Blot, In Vitro, Kinase Assay, Staining

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: (A) HeLa cells were transiently transfected with vector-only, wild-type, or S314A mutant of Bub1 followed by experiments Western blot analysis to detect Histone H2A Thr121 phosphorylation in response IR; (B) HeLa cells transiently transfected with vector-only, wild-type, S314A or S314E mutant of Bub1 followed by immunoblotting with indicated antibodies. (C) The cell survival assay after IR in HeLa cells transfected with vector, wild-type or S314A Bub1; and (D) the single cell gel electrophoresis assay to detect comet tail formation in HeLa cells transfected with vector, wild-type or S314A Bub1.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, Clonogenic Cell Survival Assay, Single Cell Gel Electrophoresis

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 1. TGFBR2 is a BUB1 kinase. (A) Radio-phosphoimages of in vitro kinase assay where His-tagged TGFBR1-WT and TGFBR1-cyto domain only (purified using Ni-NTA beads from HEK293T cells) were used as kinases. Varying concentrations of Sf9 baculovirally purified BUB1-WT (200 ng, 500 ng, 1 mg/lane) and BUB1-KD (1 mg/lane) were used as substrates. The reactions were run at 37 C for 3 h with 5–10 mCi 32p-ATP. Laemmli quenched reactions were run on SDS-PAGE gels and imaged on phosphoimager. Arrows point to the proteins as detected by protein-specific antibodies on the western blots. (B), Sf9 purified BUB1 WT (1 mg in lanes 2 &3, 2 mg in lane 4, 500 ng lane 5), BUB1-KD (1 mg), or BUB1-E (1 mg) were used as substrates while baculovirally purified GST-TGFBR2 (200 ng/lane in all lanes except lane 5 where 500 ng) was used as a kinase in in vitro kinase reactions run at 37 C for 3 h with 5–10 mCi hot-ATP. A representative radio-phosphoimage is shown. Numbers below the lanes show average fold enrichment (of BUB1-KD phosphorylation) from three different replicates. Arrows point to the proteins detected with specific antibodies in western- blots. Bub3 and BUB1-E are undetectable in radio-phosphoimages. (C) In vitro kinase assay where BUB1-WT (200 ng), GST-TGFBR2 (1 mg) along with BUB1 inhibitor 2OH-BNPP1 (10 mM) was used. The in vitro kinase reaction was performed in same conditions as in Fig. 1A and B. (D) In vitro kinase assay where purified BUB1-WT (100 ng) was used as a kinase and histone-H2A (500 ng) was used as a substrate in the presence of 10 mM 2OH- BNPP1. Radio-phosphoimage shows 2OH-BNPP1 inhibits BUB1 kinase activity in this assay.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: In Vitro, Kinase Assay, Purification, SDS Page, Western Blot, Phospho-proteomics, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 3. Phosphorylation of BUB1 at Ser318 causes reduction in interaction with TGFBR1 and SMAD2. (A) HEK293T cells were transfected with Myc- BUB1-WT, S318A, S318D mutants and HA-tagged TGFBR2, serum starved and treated for an hour with TGF-b (5 ng/mL). Lysates were made 40– 48 h post-transfections. Immunoprecipitation was performed using Myc-tag antibodies and blots were probed with TGFBR2 and Myc-tag antibodies. (B) IP for TGFBRI and then blotting for Myc in lysates from HEK293T cells transfected with Myc-BUB1-WT, S318A, S318D mutants and His-tagged TGFBR1, serum-starved, and treated with TGF-b (5 ng/mL) for 1 h. (C) IP for FLAG and then blotting for Myc in lysates from HEK293T cells transfected with BUB1-WT, S318A and S318D mutants and FL-SMAD2, serum starved and treated with TGF-b (5 ng/mL) for 1 h.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 4. BUB1 may have multiple contact points for TGF-b signaling components. (A) IP for Myc and then blotting four TGFBR2 in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutants (1–241, 242–481 and 482–723) and HA-TGFBR2, serum starved and treated with TGF-b (5 ng/mL) for 1 h. (B) IP for Myc and then blotting four His in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutants and His-TGFBR1 cytoplasmic tail, synchronized in G2/M by nocodazole. (C) IP for FLAG and then blotting for Myc in lysates from HEK293T cells transfected with BUB1 truncation mutants and FL-SMAD2, serum starved and left untreated or treated with TGF-b (5 ng/mL) for 1 h.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 5. TGFBR2 mediated phosphorylation of BUB1 reduces its interaction with TGFBR2 and SMAD2. (A) IP for Myc and then blotting for TGFBR2 in lysates from A549 cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with HA-TGFBR2. Cells were serum starved and treated with TGF-b (5 ng/mL for 1 h) before harvesting. (B) IP for Myc and then blotting for SMAD2 in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with FL-SMAD2. Cells were serum starved and treated with TGF-b (5 ng/mL for 1 hour) before harvesting.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Phospho-proteomics, Transfection, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 7. A schematic representing the proposed model with steps involved in BUB1 mediated regulation of activated TGF-b signaling complex. (i) BUB1 is recruited to TGFBR1-TGFBR2 complex in response to ligand, (ii) BUB1 participates in the recruitment of SMAD2/3 to the receptor, and (iii) TGFBR2 phosphorylates BUB1 at S318, which triggers the disassembly of the activated complex.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: