beta tubulin recombinant antibody  (Proteintech)

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora B phosphorylates serine 16 and serine 39 of BubR1 upon nocodazole treatment, which is required for K250 acetylation. ( A ) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1 marked with AurkB-binding region and phosphorylation sites. Potential phosphorylation sites (Ser16 and Ser39) by AurkB and K250 acetylation site are marked with red. N-terminus of BubR1 (1–150) binds to AurkB (results from Fig. ). (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis . They were then transfected into HeLa cells and treated with nocodazole (200 ng/ml) for 20 h. Mitotic cells were collected by shake-off, and attached cells (Attach) were employed as a control. Mitotic cell lysates, along with lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess the mitosis stage. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as loading controls. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. ( B ) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. ( C ) Effect of Ser16 or Ser39 phosphorylation in K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. ( D ) Graph showing the intensities of anti-AcK250 immunofluorescence from panel (C). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments. Number of cells: WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29 (mean ± s.e.m.). ( E ) WB analysis showing the effects of mitotic kinase inhibitors on phosphorylation of BubR1 (1–150) in unattached kinetochore (nocodazole treatment). HeLa cells were subjected to nocodazole treatment and mitotic shake-off. The mitotic lysates were then resuspended in in vitro phosphorylation buffer containing 100 μM ATP (see the “Materials and methods” section). MBP-tagged recombinant BubR1 (amino acids 1–150) was employed as the substrate. The mitotic lysate and recombinant BubR1 were incubated in the buffer for 2 h with/without indicated inhibitors (10 nM BI 2536, 2 μM reversine, 200 nM hesperadin, and 2 μM ZM447439). In parallel, the lysates were subjected to WB with anti-AurkB-pT232, -AurkB, and -β-actin. ( F ) In vitro phosphorylation assay showing that phosphorylation of S39 and S16 is AurkB-dependent. Recombinant BubR1 (amino acids 1–150) was incubated with anti-AurkB immunoprecipitate of nocodazole-arrested HeLa cells. Reactions were carried out for 2 h in the presence or absence of the AurkB inhibitor ZM447439 (2 μM). [γ- 32 P]-ATP label detects the phosphorylation of the substrate BubR1 (1–150). WB with anti-MBP and anti-AurkB in the same lysate was performed for control.

Article Snippet: Commercially sourced antibodies used in this study include anti-BubR1 (612 503; BD Biosciences), anti-α-tubulin-FITC (F2168; Upstate), anti-Centromere protein antibody (15–235; Antibodies Inc.), anti-HDAC2 (ab12169; Abcam), anti-HDAC3 (ab7030; Abcam), anti-CDC20 (A15656; ABclonal), anti-MAD2 (A11469; ABclonal), anti-Cyclin B (sc-245; Santa Cruz Biotechnology), anti-β-actin (A700-057; Bethyl), anti-CDC27 (BD 610455; BD Biosciences), anti-FLAG (F1804; Sigma–Aldrich), anti-GAPDH (#2118; Cell Signaling Technology), anti-CENP-A (ab13939;Abcam), anti-ZW10 (sc-81430; Santa Cruz Biotechnology), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-AIM-1 (BD611082; BD Biosciences), anti-Vinculin (sc-73614; Santa Cruz Biotechnology), anti-AurB-pT232 (636 102; BioLegend), anti-PLK1 (ab17057; Abcam), anti-mCherry (ab125096; Abcam), anti-Bub3 (611 730; BD Biosciences), ani-CENP-E (A15263; ABclonal), Cyclin B (sc-245; Santa Cruz Biotechnology), anti-pH3 (06–570; EMD Millipore), anti-pan-Phospho-serine (AP0932; ABclonal), and anti-Cdc25C (A12234, ABclonal).

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Recombinant, Incubation