Journal: Oncology reports

Article Title: Prexasertib increases the sensitivity of pancreatic cancer cells to gemcitabine and S‑1.

doi: 10.3892/or.2019.7421

Figure Lengend Snippet: Figure 4. (A) Effect of prexasertib, GEM + S‑1 and prexasertib + GEM + S‑1 on Chk1 (pS296, pS317, pS345), Chk1, γH2AX, and H2AX levels in SUIT‑2 cells. (B and C) Western blot analysis revealed the levels of caspase 9, caspase 3, and PARP and Bcl‑2 family. (D) Release of cytochrome c from mitochondria was detected. SUIT‑2 cells were treated for 24, 48, and 72 h either without (control) or with prexasertib, GEM + S‑1, or prexasertib + GEM + S‑1. (E) Level of expression of Bcl‑2 at 72 h normalized to that of β‑actin (Bcl‑2/β‑actin) and reported as the fold change compared with the control value. The relative band intensities of Bcl‑2 and β‑actin were quantified using densitometric analysis. Values are expressed as the mean ± SD of three independent experiments. *P<0.05 indicates a significant difference. GEM, gemcitabine; GS, GEM + S‑1 combined treatment; Chk1, checkpoint kinase 1; PARP, poly(ADP‑ribose) polymerase.

Article Snippet: Monoclonal antibodies against phosphorylated-Chk1 (Ser296) (product #90178, 1:1,000 dilution), phosphorylated-Chk1 (Ser317) (product #12302, 1:1,000 dilution), phosphorylated-Chk1 (Ser345) (product #2348, 1:1,000 dilution), Chk1 (product #2360, 1:1,000 dilution), γH2AX (Ser139) (product #9718, 1:1,000 dilution), H2AX (product #2595, 1:1,000 dilution), caspase 9 (product #9508, 1:1,000 dilution), caspase 3 (product #9665, 1:1,000 dilution), cleaved PARP (Asp214) (product #5625, 1:1,000 dilution), Mcl-1 (product #94296, 1:1,000 dilution), Bcl-2 (product #2870, 1:1,000 dilution), Bcl-xL (product #2764, 1:1,000 dilution) and Bax (product #2772, 1:1,000 dilution) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Control, Expressing

Journal: Oncology reports

Article Title: Prexasertib increases the sensitivity of pancreatic cancer cells to gemcitabine and S‑1.

doi: 10.3892/or.2019.7421

Figure Lengend Snippet: Figure 5. Effects of Chk1‑specific siRNA on combination treatment with GEM + S‑1 (GS). (A) SUIT‑2 cells were transfected with vehicle, non‑silencing siRNA (Control‑si) and Chk1‑specific siRNA (Chk1‑si). After transfection, the levels of expression of Chk1 and β‑actin were analyzed by western blotting. (B) SUIT‑2 cells were treated with GEM + S‑1 (GS) for 24 h after transfection of Control‑si or Chk1‑si. The extent of apoptosis was quantified by Cell Death ELISA. Values shown are the mean ± SD. *P<0.05 indicates a significant difference. (C) After staining of the cells with Hoechst 33342, morphological changes were analyzed under a fluorescence microscope, or assessed by phase contrast microscopy. The scale bar indicates 10 µm. (D) Western blot analysis shows changes in the expression of apoptosis‑related factors in SUIT‑2 cells treated with GEM + S‑1 (GS) for 24 h after transfection of Control‑si or Chk1‑si. (E) Level of expression of Bcl‑2 normalized to that of β‑actin (Bcl‑2/β‑actin) and reported as the fold change compared with the control value. The relative band intensities of Bcl‑2 and β‑actin were quantified using densitometric analysis. Values are expressed as the mean ± SD of three independent experiments. *P<0.05 indicates a significant difference. NS, not significant; GEM, gemcitabine; GS, GEM + S‑1 combined treatment; Chk1, checkpoint kinase 1.

Article Snippet: Monoclonal antibodies against phosphorylated-Chk1 (Ser296) (product #90178, 1:1,000 dilution), phosphorylated-Chk1 (Ser317) (product #12302, 1:1,000 dilution), phosphorylated-Chk1 (Ser345) (product #2348, 1:1,000 dilution), Chk1 (product #2360, 1:1,000 dilution), γH2AX (Ser139) (product #9718, 1:1,000 dilution), H2AX (product #2595, 1:1,000 dilution), caspase 9 (product #9508, 1:1,000 dilution), caspase 3 (product #9665, 1:1,000 dilution), cleaved PARP (Asp214) (product #5625, 1:1,000 dilution), Mcl-1 (product #94296, 1:1,000 dilution), Bcl-2 (product #2870, 1:1,000 dilution), Bcl-xL (product #2764, 1:1,000 dilution) and Bax (product #2772, 1:1,000 dilution) were purchased from Cell Signaling Technology.

Techniques: Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Microscopy, Control

Journal: Cell Reports

Article Title: Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway

doi: 10.1016/j.celrep.2023.112721

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-pCHK1 (Ser345), (133D3) , Cell signaling technology , Cat# 2348; RRID: AB331212.

Techniques: Virus, Recombinant, DNA Sequencing, Knock-In, Software, Microscopy

Journal: bioRxiv

Article Title: Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM

doi: 10.1101/2024.08.26.609797

Figure Lengend Snippet: ( A ) GBM cell viability of BIA and cisplatin combination treatment. Cisplatin doses are indicated in x-axis, BIA remained at a constant concentration of 1 μM (5 days exposure). Mean and standard deviation are shown, n=3/group. ( B ) Dose-response matrix showing inhibition percentage of BIA and cisplatin combinations at various concentrations for 5 days using SynergyFinder 3.0. G9-PCDH cells were treated, and viability analyzed as indicated in the cell viability assay section (see Materials and Methods). ( C ) ZIP method synergy score of BIA and cisplatin combinations. The overall average δ -score is indicated on top of the chart. The dose combinations showing an increased likelihood of synergy are highlighted. ( D ) Immunofluorescence staining of γH2AX (green) and nuclei (blue) in G9-PCDH cells treated with 1 μM of cisplatin and/or BIA, for 72 hours. Representative image of 3 pictures per condition. Pictures taken at 40x, scale bar=20 μm. ( E ) Quantification of γH2AX foci from ( D ) using ImageJ. ( F ) Western blot of G9-PCDH cells treated with 1 μM of cisplatin and/or BIA, for 72 hours, probing for the p-CHK1 (Ser345), total CHK1 and H2AX (Ser139) proteins. GAPDH was used as loading control. Representative image from triplicate experiments.

Article Snippet: Primary antibodies used were incubated in the cold under shaking overnight: anti-pCHK1 (Ser345) (Cell Signaling Technologies) 1:100, anti-CHK1 (Cell Signaling Technologies) 1:1000, anti-pH2AX (Ser139) (Cell Signaling Technologies) 1:1000, anti-MFSD2A (Proteintech) 1:500, anti-CAV1 (Proteintech) 1:1000, anti-CD144 (VE-cadherin) (Thermo Fisher Scientific) 1:1000, anti-β-actin (Cell Signaling Technologies) 1:2000.

Techniques: Concentration Assay, Standard Deviation, Inhibition, Viability Assay, Immunofluorescence, Staining, Western Blot, Control

Journal: bioRxiv

Article Title: Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast

doi: 10.1101/2023.03.06.531248

Figure Lengend Snippet: ( A ) Sensitivities of the four previously reported RPA mutants to HU and MMS were examined by spot assay. A series of five-fold dilutions of the logarithmically growing cells were spotted on YE6S plates or plates containing HU or MMS. The plates were incubated at 30°C for 3 days and then photographed. Wild-type (TK48) cells and the checkpoint mutants rad3 Δ (NR1826), cds1 Δ (GBY191), and chk1 Δ (TK197) were included as controls. ( B ) Phosphorylation of Mrc1 by Rad3 was unaffected or moderately reduced in the four RPA mutants. Wild type and the mutant cells used in A were treated with (+) or without (−) 15 mM HU for 3 h. Phosphorylation of Mrc1 (upper panel) was examined by Western blotting of whole cell lysates made from the TCA-fixed cells after SDS-PAGE using the phospho-specific antibody. The same blot was stripped and reprobed with anti-Mrc1 antibodies (middle panel). A section of the Ponceau S-stained membrane is shown for loading control (bottom panel). The phosphorylation bands were quantified, and the intensities relative to the HU-treated wild-type cells are shown at the bottom. ( C ) The Western blotting shown in A was repeated three times and the quantitation results are shown in percentages. Error bars represent the means and SDs of the triplicates. Blue and brown columns indicate before and after HU treatment, respectively. ( D ) Phosphorylation of Cds1 by Rad3 was increased or moderately reduced in the four RPA mutants. Wild type and the indicated mutant cells were treated with HU as in B. Cds1 was IPed and analyzed by Western blotting using an anti-HA antibody (bottom panel). The same membrane was stripped and then blotted with the phospho-specific antibody (upper panel). The phosphorylation bands were quantified and relative intensities are shown at the bottom. ( E ) The experiments in D were repeated three times and the quantitation results are shown. ( F ) Chk1 phosphorylation was examined in wild-type and the mutant cells treated with (+) or without (−) 0.001% MMS for 90 min. The whole cell lysates made by the TCA method were analyzed by SDS-PAGE followed by Western blotting with anti-HA antibody. ( G ) Quantitation results from three separate blots as in F are shown in ratios of phosphorylated Chk1 vs total Chk1. ( H ) Chk1 phosphorylation was examined by Western blotting using the phospho-specific antibody. Wild type and the indicated mutant cells were treated MMS as in F. Chk1 was IPed and then analyzed by Western blotting using the antibody against Chk1-pS345 (top panel). The same membrane was stripped and blotted with an anti-HA antibody (bottom panel). The relative intensities of the Chk1-pS345 bands were quantified, normalized with that of Chk1 bands, and shown in percentages. ( I ) Quantitation results from three repeats of H are shown. ( J ) Sensitivities of the RPA mutants to acute HU treatment were determined by spot assay as described in Materials and Methods. ( K ) Specificity of the antibody against Ssb1. S. pombe expressing with untagged or tagged Ssb1 were lysed and analyzed by Western blotting using the anti-Ssb1 antibody or pre-immune antiserum (upper panels). The Ponceau S-stained membrane is shown for loading (middle panels). The same blot was stripped and reblotted sequentially with anti-HA and then anti-Flag antibodies to confirm the tagged Ssb1 (lower two panels). ( L ) Ssb1 levels were examined by Western blotting in wild type and the indicated mutant cells before (−) or after (+) HU treatment (upper panels). Loading is shown by Ponceau S staining. The relative levels of Ssb1 are shown at the bottom. The lower panel is the quantitation results of three separate blots.

Article Snippet: The custom antibody against phosphorylated Chk1-Ser 345 used in this study was generated in rabbits and purified by Bethyl Laboratories (Montgomery, TX).

Techniques: Spot Test, Incubation, Phospho-proteomics, Mutagenesis, Western Blot, SDS Page, Staining, Membrane, Control, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast

doi: 10.1101/2023.03.06.531248

Figure Lengend Snippet: ( A ) Rad3-dependent Mrc1 phosphorylation in the thirteen ssb1 mutants was examined as in (left panels). Quantitation results from three repeats are shown on the right. ( B ) Rad3-dependent Cds1 phosphorylation in the ssb1 mutants was examined (left panels), repeated three times, and the quantitation results are shown on the right. ( C ) Rad3-dependent Chk1 phosphorylation in the ssb1 mutants treated with MMS was examined by Western blotting using the phospho-specific antibody as in . The quantitation results are shown on the right. ( D ) The Chk1 phosphorylation in the MMS-treated ssb1 mutants was examined by the mobility shift assay as in . The quantitation results are shown on the right.

Article Snippet: The custom antibody against phosphorylated Chk1-Ser 345 used in this study was generated in rabbits and purified by Bethyl Laboratories (Montgomery, TX).

Techniques: Phospho-proteomics, Quantitation Assay, Western Blot, Mobility Shift

Journal: bioRxiv

Article Title: Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast

doi: 10.1101/2023.03.06.531248

Figure Lengend Snippet: ( A ) Diagram of Ssb1 and the relative positions of amino acid changes caused by the mutations. The four DNA binding domains F, A, B, and C are shown in blue. The intensively screened N-terminal region containing the F domain is enlarged. Dots indicate the relative locations of the mutated amino acid residues. While the yellow dots indicate the mutations that were identified once, the purple dots are those that were identified at least two times in separate mutants. The red dot indicates ssb1-R46E mutation that is analogous to the budding yeast rfc1-t11 in S. pombe . ( B ) The cell growth, drug sensitivities, Ssb1 levels, and checkpoint defects of the twenty-five primary ssb1 mutants identified in this study. The number of the primary mutants and their mutations are shown in the 1 st and 2 nd columns from the left, respectively. Numbers in parentheses indicate the times the mutants were independently screened. Asterisks in the 3 rd column indicate the relative cell growth status estimated on YE6S plates in the spot assays ( and S4A). Relative rensitivities to chronic ( and S4A) and acute HU treatment ( and S4B) determined by spot assay are shown by the asterisks in the 4 th and 5 th columns, respectively. R: resistance; UD: undetectable or minimal sensitivity. Relative Ssb1 levels in logarithmically growing cells were shown in the 7 th column. The numbers in parentheses are SD values of three repeats. Similarly, phosphorylation Mrc1 and Cds1 in HU are shown in the 8 th and 9 th columns, respectively. Chk1 phosphorylations determined by phospho-specific antibody and the mobility shift assay are shown in the 10 th and 11 th columns, respectively. The numbers in the highlighted twelve mutants in the 11 th column (lower part) were from a separate experiment. The ratio of pChk1/total Chk1 in wild-type control for the twelve mutants is 43.1 ± 4.7 (n=3). The six primary mutants selected for further characterization are marked by the dots on the left. The two mutants with confirmed partial DRC defects are marked by the green dots. The red dots indicate the mutants whose “checkpoint defects” are caused by secondary mutations. Brown dots are those with largely intact checkpoints.

Article Snippet: The custom antibody against phosphorylated Chk1-Ser 345 used in this study was generated in rabbits and purified by Bethyl Laboratories (Montgomery, TX).

Techniques: Binding Assay, Mutagenesis, Spot Test, Phospho-proteomics, Mobility Shift, Control

Journal: bioRxiv

Article Title: Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast

doi: 10.1101/2023.03.06.531248

Figure Lengend Snippet: ( A ) Six primary ssb1 mutants with more prominant checkpoint defects were selected. Their mutations were confirmed by integrating at the genomic locus in a wild-type strain. Drug sensitivities of the integrants referred to as ssb1-1 , ssb1-7 , ssb1-10 , ssb1-17 , ssb1-19 , and ssb1-24 were examined by spot assay and compared with their corresponding primary mutants. Dashed lines indicate the discontinuity. Phosphorylation of Mrc1 ( B) and Cds1 ( C ) in the six mutant integrants was examined as in and . Quantitation results from three independent blots are shown in Fig. S8A and B, respectively. Chk1 phosphorylation in the six integrants was examined as in and by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in Fig. S8C and D, respectively. Rad9 phosphorylation was examined in IPed samples using the phospho-specific antibody in the presence of HU ( F) or MMS (G ). Quantitation results are shown in Fig. S8E and F, respectively.

Article Snippet: The custom antibody against phosphorylated Chk1-Ser 345 used in this study was generated in rabbits and purified by Bethyl Laboratories (Montgomery, TX).

Techniques: Spot Test, Phospho-proteomics, Mutagenesis, Quantitation Assay, Mobility Shift

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway

doi: 10.1074/jbc.m506460200

Figure Lengend Snippet: FIGURE 1. Human Claspin is cleaved by a caspase during apoptosis. A, cleavage of Claspin in a cell-free system. Caspase activation was induced in a system made from equal volumes of HeLa cytosolic (S100) and nuclear extracts by incu- bation with cytochrome c. B, cleavage of Claspin in a cell-free system is blocked by caspase inhibitors. HeLa extracts were incubated with cytochrome c (cyt c) and either Ac-DEVD-CHO (D) or Z-VAD-fmk (Z) as indicated. Samples removed at the start of the incubation or after 8 h were analyzed by West- ern blotting with antibody to Claspin, PARP, caspase-7, or Chk1. C, caspase-dependent cleav- ageofClaspininHeLacells.Cellsweretreatedwith tumor necrosis factor- (TNF) and cycloheximide (CHX) together with Ac-DEVD-CHO as shown. D, caspase-dependent cleavage of Claspin in Jurkat cells. Cells were treated with etoposide with or without 10 M Z-VAD-fmk for the times shown. In each experiment, samples were removed after the times shown and were analyzed by Western blot- ting with antibody against Claspin, PARP, caspase-7 (Casp 7), Chk1, the Claspin C-terminal fragment (ClaspinCT), Chk1 phosphorylated at Ser345 (pChk1), or ATR as indicated. The migration positions of Claspin forms with apparent molecu- lar masses of 250 kDa (p250), 220 kDa (p220), and 30 kDa (p30) are indicated by arrows. Nonspecific bands reacting with antibody are labeled with an asterisk.

Article Snippet: The commercial antibodies used were as follows: rabbit anti-Claspin (Ab73, Bethyl Laboratories); goat anti-ATR (FRP1; N-19), mouse monoclonal anti-Chk1 (for Western blotting; G4), rabbit anti-caspase-3 (for Western blotting; H-277), goat anti-caspase-3 p11 (for immunodepletions; K-19), goat anticaspase-7 p20 (N-17), and rabbit anti-caspase-9 (for depletions; H-83) (Santa Cruz Biotechnology, Inc.); rabbit anti-phospho-Ser345/Ser317/ Ser296 Chk1 (Cell Signaling Technology); rabbit anti-poly(ADP-ribose) polymerase (PARP3; AHP427, Serotec); and mouse monoclonal anticaspase-8 and rabbit anti-caspase-9 (for Western blotting) (Pharmingen).

Techniques: Activation Assay, Incubation, Western Blot, Migration, Labeling

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway

doi: 10.1074/jbc.m506460200

Figure Lengend Snippet: FIGURE 4. Phosphorylation of Claspin and inter- action with Chk1. A, phosphorylation of Claspin fragments in a human cell-free system. In vitro translated 35S-labeled Claspin-(679–1332), Claspin- (679–1072), and Claspin-(1073–1332) (C-terminal fragment (CTF)) were incubated for 30 min at 30 °C in a mixture of HeLa cytosolic (S100) and nuclear extracts supplemented with 50 ng/l (dA)70(dT)70 (poly(dA/dT)70), 10 M okadaic acid (OA), 5 mM caf- feine (caff), 5 M staurosporine (stau), or 20 M Ro 31-8220 (Ro) as indicated. Samples were analyzed by SDS-PAGE and autoradiography. B, interaction ofChk1withphosphorylatedClaspin.Invitrotrans- lated 35S-labeled Claspin-(679–1332), Claspin-(679– 1072), and Claspin-(1073–1332) were incubated for 30 min at 30 °C in a mixture of HeLa cytosolic (S100) and nuclear extracts supplemented with 50 ng/l (dA)70(dT)70 and 10 M okadaic acid as indi- cated. Immunoprecipitations with anti-Chk1 (Chk1 IP) or control sheep (Control IP) antibody were carried out, and bound proteins were ana- lyzedbyblottingforChk1withmousemonoclonal anti-Chk1 antibody or by autoradiography for Claspin proteins.

Article Snippet: The commercial antibodies used were as follows: rabbit anti-Claspin (Ab73, Bethyl Laboratories); goat anti-ATR (FRP1; N-19), mouse monoclonal anti-Chk1 (for Western blotting; G4), rabbit anti-caspase-3 (for Western blotting; H-277), goat anti-caspase-3 p11 (for immunodepletions; K-19), goat anticaspase-7 p20 (N-17), and rabbit anti-caspase-9 (for depletions; H-83) (Santa Cruz Biotechnology, Inc.); rabbit anti-phospho-Ser345/Ser317/ Ser296 Chk1 (Cell Signaling Technology); rabbit anti-poly(ADP-ribose) polymerase (PARP3; AHP427, Serotec); and mouse monoclonal anticaspase-8 and rabbit anti-caspase-9 (for Western blotting) (Pharmingen).

Techniques: Phospho-proteomics, In Vitro, Labeling, Incubation, SDS Page, Autoradiography, Control

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway

doi: 10.1074/jbc.m506460200

Figure Lengend Snippet: FIGURE 5. Caspase-mediated cleavage of Claspin separates a C-terminal DNA-inter- acting domain from the Chk1-binding domain. A, interaction of Claspin with double- stranded oligonucleotides. In vitro translated 35S-labeled Claspin-(679–1332) was incu- bated for 30 min at 30 °C in a mixture of HeLa cytosolic (S100) and nuclear extracts supplemented with 10 M okadaic acid (OA) and 50 ng/l (dA)70(dT)70 (poly(dA/dT)70), either non-biotinylated or biotinylated at the 5- or 3-end of the poly(dA) strand as indicated. Proteins precipitated with streptavidin beads or in the total extract were ana- lyzed by SDS-PAGE and autoradiography. B, interaction of Claspin with double-stranded oligonucleotides requires a C-terminal domain. Extract was incubated with in vitro trans- lated 35S-labeled full-length Claspin, its large N-terminal fragment (NTF; amino acids 1–1072), or its smaller C-terminal fragment (CTF; amino acids 1073–1332) together with okadaic acid and (dA)70(dT)70, either non-biotinylated or 5-biotinylated. Proteins in the total extract or bound to streptavidin beads were analyzed by SDS-PAGE and autora- diography. C, inhibition of Chk1 phosphorylation by Claspin-(1073–1332). HeLa extract was incubated for 30 min at 30 °C with okadaic acid plus (dA)70(dT)70 and, at the con- centrations shown, the His6-tagged C-terminal fragment (Claspin-(1073–1332)) or His6- C287A, an inactive caspase-9 mutant, as a control (Ctrl). Samples were analyzed by SDS- PAGE and Western blotting with anti-Chk1 and anti-phospho-Ser345 Chk1 antibodies.

Article Snippet: The commercial antibodies used were as follows: rabbit anti-Claspin (Ab73, Bethyl Laboratories); goat anti-ATR (FRP1; N-19), mouse monoclonal anti-Chk1 (for Western blotting; G4), rabbit anti-caspase-3 (for Western blotting; H-277), goat anti-caspase-3 p11 (for immunodepletions; K-19), goat anticaspase-7 p20 (N-17), and rabbit anti-caspase-9 (for depletions; H-83) (Santa Cruz Biotechnology, Inc.); rabbit anti-phospho-Ser345/Ser317/ Ser296 Chk1 (Cell Signaling Technology); rabbit anti-poly(ADP-ribose) polymerase (PARP3; AHP427, Serotec); and mouse monoclonal anticaspase-8 and rabbit anti-caspase-9 (for Western blotting) (Pharmingen).

Techniques: Binding Assay, In Vitro, Labeling, SDS Page, Autoradiography, Incubation, Inhibition, Phospho-proteomics, Mutagenesis, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway

doi: 10.1074/jbc.m506460200

Figure Lengend Snippet: FIGURE 6. Model of the effect of Claspin cleav- age by caspase-7. A, caspase-7 (Casp7) cleaves Claspin after Asp1072, generating a large N-termi- nal fragment (NTF) containing a Chk1-binding domain (CKBD) that includes a motif phosphoryl- ated at two sites, Thr916 and Ser945, and a smaller C-terminal fragment (CTF) required for association with DNA. aa, amino acids. B, phosphorylation and activation of Chk1 by ATR in response to DNA damage or during DNA replication require recruit- ment of Chk1 to DNA by Claspin. With prolonged DNA damage or DNA replication arrest, activation of caspase-7 results in proteolytic cleavage of Claspin, dephosphorylation of Chk1, inactivation of the checkpoint signal, and promotion of apo- ptosis. Activated forms of Chk1 and ATR are indi- cated by asterisks.

Article Snippet: The commercial antibodies used were as follows: rabbit anti-Claspin (Ab73, Bethyl Laboratories); goat anti-ATR (FRP1; N-19), mouse monoclonal anti-Chk1 (for Western blotting; G4), rabbit anti-caspase-3 (for Western blotting; H-277), goat anti-caspase-3 p11 (for immunodepletions; K-19), goat anticaspase-7 p20 (N-17), and rabbit anti-caspase-9 (for depletions; H-83) (Santa Cruz Biotechnology, Inc.); rabbit anti-phospho-Ser345/Ser317/ Ser296 Chk1 (Cell Signaling Technology); rabbit anti-poly(ADP-ribose) polymerase (PARP3; AHP427, Serotec); and mouse monoclonal anticaspase-8 and rabbit anti-caspase-9 (for Western blotting) (Pharmingen).

Techniques: Binding Assay, Phospho-proteomics, Activation Assay, De-Phosphorylation Assay