Anti-TopBP1 antibody

• IP

Supplier Data

Immunoprecipitation - Anti-TopBP1 antibody (AB2402)

Samples : Whole cell lysate (1.0 mg per IP reaction; 20% of IP loaded) from HeLa cells prepared using NETN lysis buffer.

Antibodies : ab2409 used for IP at 3 μg per reaction.

Two lots tested (Lanes 1 and 2).

Lane 3 is Control IgG.

For blotting immunoprecipitated TopBP1, ab2409 was used at 0.4 μg/ml. Detection : Chemiluminescence with an exposure time of 10 seconds.

All lanes:

Immunoprecipitation - Anti-TopBP1 antibody (ab2402)

Predicted band size: 170 kDa

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• WB

Supplier Data

Western blot - Anti-TopBP1 antibody (AB2402)

Lysates prepared using NETN lysis buffer.

All lanes:

Anti-Cytokeratin 10 antibody [LHP1] (<a href='/ko/products/unavailable/cytokeratin-10-antibody-lhp1-ab2409'>ab2409</a>) at 0.1 µg/mL

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 15 µg

Lane 2:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 15 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 15 µg

Predicted band size: 170 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-TopBP1 antibody (AB2402)

Lysates prepared using NETN lysis buffer.

All lanes:

Anti-Cytokeratin 10 antibody [LHP1] (<a href='/ko/products/unavailable/cytokeratin-10-antibody-lhp1-ab2409'>ab2409</a>) at 0.1 µg/mL

Lane 1:

TCMK-1 (Mouse kidney epithelial cell line) whole cell lysate at 50 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 50 µg

Predicted band size: 170 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-TopBP1 antibody (AB2402)

Western Blotting using Anti-TopBP1 antibody, ab2402. Publication image from Mooser, C. et al., 2020, Nat Commun, 31913317. Legend direct from paper.

A conserved TOPBP1 phospho-interaction motif in Treacle.a HA-immunoprecipitations from 293FT cells transfected with the indicated HA-tagged Treacle variants. b TOPBP1 localization 2 h after I-Ppo1 transfection in GFP-Treacle-expressing cell lines after treatment with control siRNA (left panel) and Treacle siRNA (right panel). c Quantification of the experiment in b, mock : siCtrl (n = 301) versus siTreacle (n = 290), WT : siCtrl (n = 226) versus siTreacle (n = 242), S1236A : siCtrl (n = 75) versus siTreacle (n = 79), S1227A/S1228A : siCtrl (n = 169) versus siTreacle (n = 177) and S1227A/S1228A/S1236A : siCtrl (n = 77) versus siTreacle (n = 69) cells. d Quantification of nucleolar EU incorporation after I-Ppo1 transfection in GFP-Treacle-expressing cell lines after treatment with control siRNA and Treacle siRNA. WT : siCtrl (n = 216) versus siTreacle (n = 200), S1236A : siCtrl (n = 224) versus siTreacle (n = 224), S1227A/S1228A : siCtrl (n = 222) versus siTreacle (n = 227) and S1227A/S1228A/S1236A : siCtrl (n = 229) versus siTreacle (n = 232) cells. c, d Statistical significance was calculated using a one-way analysis of variance and Sidak’s multiple comparison test. Bars represent mean ± S.D. All scale bars = 10 µm. Source data are provided as a Source Data file.

false

• WB

CiteAb

Western blot - Anti-TopBP1 antibody (AB2402)

Western Blotting using Anti-TopBP1 antibody, ab2402. Publication image from Mooser, C. et al., 2020, Nat Commun, 31913317. Legend direct from paper.

TOPBP1 recruitment in response to rDNA breaks.a Timecourse of TOPBP1 localization in GFP-Treacle expressing U2OS cells after I-Ppo1 transfection. b TOPBP1 localization 2 h after I-Ppo1 transfection in NBS1δN cells and NBS1δN cells complemented with wild-type and mutant NBS1-mNG (percentage of cells with > 2 TOPBP1 caps are indicated; one of two experiments is shown). c TOPBP1 localization 2 h after I-Ppo1 transfection in ATMi- and ATRi-treated cells versus vehicle (DMSO)-treated cells (percentage of cells with TOPBP1 foci inside of the nucleoli are indicated; one of two experiments is shown). d Quantification of cells with TOPBP1 caps after ATMi and ATRi treatment versus vehicle (DMSO) treatment (red bars represent mean). e Quantification of nucleolar EU incorporation after I-Ppo1 H98A (n = 692) and I-Ppo1 WT (n = 649) transfection in control U2OS and after I-Ppo1 H98A (n = 450) and I-Ppo1 WT (n = 521) transfection in siTOPBP1-treated cells (boxes represent the median with 25–75 percentile range and whiskers represent the 5–95 percentile range. Data points outside of this range are shown individually). f Endogenous Treacle localization 2 h after I-Ppo1 transfection in control siRNA and TOPBP1 siRNA transfected cells (percentage of cells with > 2 Treacle caps are indicated; one of two experiments is shown). g TOPBP1 localization in Treacle-depleted U2OS cells and control cells 2 h after I-Ppo1 transfection (percentage of cells with > 2 TOPBP1 caps are indicated; one of two experiments is shown). h HA-immunoprecipitations from 293FT cells transfected with the indicated HA-tagged Treacle variants, in the presence or absence of ATMi and ATRi and with and without IR treatment as indicated. STTT mutated Treacle : S171A, T173A, T203A, T210A. All scalebars = 10 µm. Source data are provided as a Source Data file.

false

• WB

CiteAb

Western blot - Anti-TopBP1 antibody (AB2402)

Western Blotting using Anti-TopBP1 antibody, ab2402. Publication image from Mooser, C. et al., 2020, Nat Commun, 31913317. Legend direct from paper.

Characterization of the TOPBP1–Treacle interaction.a Schematic showing the layout of conserved domains and motifs in Treacle as well as HA-tagged deletion constructs of Treacle, lacking either the N-terminal region (F1), both the N-terminal and the central repeat region (F2), the central repeat region (F3), the C-terminal region (F4) or both the central repeat and the C-terminal region (F5). HA-immunoprecipitation of full-length and five deletion constructs depicted in a. c Schematic showing the layout of conserved domains and motifs in TOPBP1. Names of known TOPBP1-binding partners are shown below the domains they interact with and phospho-interacting Lys residues in the three BRCT tandems are highlighted. AAD : ATR-activating domain. d GFP-pulldowns from 293FT cells transfected with the indicated GFP-tagged TOPBP1 constructs. Source data are provided as a Source Data file.

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