anti phosphorylated chk1 ser 345 Search Results


86
Millipore phosphorylated ser 345 chk1
Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of <t>Chk1</t> phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.
Phosphorylated Ser 345 Chk1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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86
Abcam phosphorylated ser 345 chk1
Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of <t>Chk1</t> phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.
Phosphorylated Ser 345 Chk1, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/phosphorylated ser 345 chk1/product/Abcam
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86
Cell Signaling Technology Inc phosphorylated chk1 ser 345
Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of <t>Chk1</t> phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.
Phosphorylated Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/phosphorylated chk1 ser 345/product/Cell Signaling Technology Inc
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86
Santa Cruz Biotechnology rabbit anti chk1 phosphorylated ser 345
(A) Phosphorylation of <t>Chk1</t> in HeLa cell extracts. HeLa cytoplasmic extract (S100), nuclear extract (HNE) and a mixture of equal volumes of the two (HNE/S100) were incubated at 30 °C for 30 min with or without an ATP-regenerating system, 10 μM OA and 50 ng/μl poly(dA/dT)70, as indicated. One sample contained 10 mM EDTA to block kinase activity. Western blots of protein separated by gel electrophoresis were probed with antibodies to Chk1 (anti-Chk1) or the phosphorylated Ser345 site in Chk1 (α-p345). (B) Time course of Chk1 phosphorylation. The extract was incubated with OA and poly(dA/dT)70 for the times shown and blotted with antibodies to Chk1 (anti-Chk1). (C) Chk1 is phosphorylated on Ser345 in response to dsDNA, not single-stranded DNA. The extract was incubated with OA and poly(dA/dT)70, poly(dA)70, poly(dT)70 or poly(dA/dT)40 as indicated, then Western blotted with either anti-Chk1 or α-p345. (D) Phosphorylation of Chk1 on Ser296 and Ser317 in response to oligonucleotides. The extract was incubated with OA and poly(dA/dT)70 as indicated and Western blotted with antibodies to Chk1 (anti-Chk1) and the phosphorylated Ser296 (α-p296) and Ser317 (α-p317) sites in Chk1.
Rabbit Anti Chk1 Phosphorylated Ser 345, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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86
Abcam phosphorylated chk1 345
(A) Phosphorylation of <t>Chk1</t> in HeLa cell extracts. HeLa cytoplasmic extract (S100), nuclear extract (HNE) and a mixture of equal volumes of the two (HNE/S100) were incubated at 30 °C for 30 min with or without an ATP-regenerating system, 10 μM OA and 50 ng/μl poly(dA/dT)70, as indicated. One sample contained 10 mM EDTA to block kinase activity. Western blots of protein separated by gel electrophoresis were probed with antibodies to Chk1 (anti-Chk1) or the phosphorylated Ser345 site in Chk1 (α-p345). (B) Time course of Chk1 phosphorylation. The extract was incubated with OA and poly(dA/dT)70 for the times shown and blotted with antibodies to Chk1 (anti-Chk1). (C) Chk1 is phosphorylated on Ser345 in response to dsDNA, not single-stranded DNA. The extract was incubated with OA and poly(dA/dT)70, poly(dA)70, poly(dT)70 or poly(dA/dT)40 as indicated, then Western blotted with either anti-Chk1 or α-p345. (D) Phosphorylation of Chk1 on Ser296 and Ser317 in response to oligonucleotides. The extract was incubated with OA and poly(dA/dT)70 as indicated and Western blotted with antibodies to Chk1 (anti-Chk1) and the phosphorylated Ser296 (α-p296) and Ser317 (α-p317) sites in Chk1.
Phosphorylated Chk1 345, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of Chk1 phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.

Journal: The Journal of Cell Biology

Article Title: DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

doi: 10.1083/jcb.201207066

Figure Lengend Snippet: Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of Chk1 phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.

Article Snippet: We used primary antibodies against Pol η (Abcam), FlagM2 (Sigma-Aldrich), phosphorylated Ser 345-Chk1 (Cell Signaling Technology), Chk1 (Santa Cruz Biotechnology, Inc.), PCNA (Abcam), RPA (EMD Millipore), ORC2 (MBL), FANCD2 (Santa Cruz Biotechnology, Inc.), α-tubulin (Sigma-Aldrich), actin (EMD Millipore), vinculin (Abcam), caspase-3 (Cell Signaling Technology), ATR (Cell Signaling Technology), SMC2 (Abcam), and MCM7 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Microscopy, Software, MANN-WHITNEY, Immunodetection, Positive Control, Stable Transfection, Western Blot, Fractionation, Mutagenesis

Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of Chk1 phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.

Journal: The Journal of Cell Biology

Article Title: DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

doi: 10.1083/jcb.201207066

Figure Lengend Snippet: Analysis of the replication stress in Pol η–deficient cells and Dead Pol η–expressing cells. (A) Left and middle: mock-depleted (ShCtrl) and Pol η–depleted (ShPolη) U2OS cells were randomly acquired with wide-field microscopy ( n > 70 cells) in three independent experiments. Quantification of RPA-positive nuclei (right) and number of RPA foci per nucleus (middle) was performed in PCNA foci–positive nuclei (S phase). For the quantification of RPA-positive cells, the p-value was determined with the t-test (*, P = 0.019; standard deviations are indicated by error bars. The number of RPA foci per nucleus was counted with ImageJ software ( n = 234; National Institutes of Health). The p-value was determined with the non-parametric Mann-Whitney test (***, P < 0.005). Right: example of images from ShCtrl and ShPolη cells immunostained with anti PCNA (red), anti RPA (green), and DAPI (blue). Triton preextraction was performed before fixation. Bar, 10 µM. (B) Analysis of Chk1 phosphorylation in Pol η–depleted U2OS cells. Anti-Pol η, P-Chk1(ser 345), and Chk1 immunodetection on whole extracts from U2OS cells treated with UV (20J/m 2 , 6 h) as positive control for Chk1 phosphorylation, U2OS cells untreated and U2OS cells mock depleted (ShCtrl), or Pol η–depleted (ShPolη). Chk1 serves here as a loading control. (C) Extracts from XPV cells, XPV cells stably complemented with Pol η WT (XPV+Polη WT), or the Dead form of Pol η (XPV+Polη Dead1 and XPV+Polη Dead2) were fractionated and the soluble fractions were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and α-tubulin as loading control. (D) The chromatin fraction from extracts described in C were analyzed by immunoblotting for RPA level into the chromatin. ORC2 was used as a loading and fractionation control. (E) Requirement of PIP and UBZ domains of Dead Pol η for its ability to activate the replication checkpoint. Extracts from XPV cells, XPV cells stably complemented with wild-type Pol η (XPV+WT Polη), Dead Pol η (XPV+Dead Pol η), D652A-Dead Pol η (XPV+Flag-D652A-Dead Pol η), ΔPIP-Dead Pol η (XPV+Flag-ΔPIP-Dead Pol η), or the double mutant D652A-ΔPIP Dead Pol η (XPV+Flag-D652A-ΔPIP-Dead Pol η) were analyzed by immunoblotting for the detection of Pol η, P-Chk1(ser 345), Chk1, and actin as loading control. The detection of Pol η was performed with Pol η antibodies (Abcam) in extracts from XPV+WT Polη, XPV+Dead Pol η, and XPV+Flag-D652A-Dead Pol η while the XPV+Flag-ΔPIP-Dead Pol η and the XPV+Flag-D652A-ΔPIP-Dead extracts were blotted with the FlagM2 antibody because the ΔPIP mutants are not recognized by the Pol η antibody (Abcam). Extracts from XPV cells treated with UV (20J/m 2 , 6 h) serves as positive control for Chk1 phosphorylation.

Article Snippet: We used primary antibodies against Pol η (Abcam), FlagM2 (Sigma-Aldrich), phosphorylated Ser 345-Chk1 (Cell Signaling Technology), Chk1 (Santa Cruz Biotechnology, Inc.), PCNA (Abcam), RPA (EMD Millipore), ORC2 (MBL), FANCD2 (Santa Cruz Biotechnology, Inc.), α-tubulin (Sigma-Aldrich), actin (EMD Millipore), vinculin (Abcam), caspase-3 (Cell Signaling Technology), ATR (Cell Signaling Technology), SMC2 (Abcam), and MCM7 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Microscopy, Software, MANN-WHITNEY, Immunodetection, Positive Control, Stable Transfection, Western Blot, Fractionation, Mutagenesis

(A) Phosphorylation of Chk1 in HeLa cell extracts. HeLa cytoplasmic extract (S100), nuclear extract (HNE) and a mixture of equal volumes of the two (HNE/S100) were incubated at 30 °C for 30 min with or without an ATP-regenerating system, 10 μM OA and 50 ng/μl poly(dA/dT)70, as indicated. One sample contained 10 mM EDTA to block kinase activity. Western blots of protein separated by gel electrophoresis were probed with antibodies to Chk1 (anti-Chk1) or the phosphorylated Ser345 site in Chk1 (α-p345). (B) Time course of Chk1 phosphorylation. The extract was incubated with OA and poly(dA/dT)70 for the times shown and blotted with antibodies to Chk1 (anti-Chk1). (C) Chk1 is phosphorylated on Ser345 in response to dsDNA, not single-stranded DNA. The extract was incubated with OA and poly(dA/dT)70, poly(dA)70, poly(dT)70 or poly(dA/dT)40 as indicated, then Western blotted with either anti-Chk1 or α-p345. (D) Phosphorylation of Chk1 on Ser296 and Ser317 in response to oligonucleotides. The extract was incubated with OA and poly(dA/dT)70 as indicated and Western blotted with antibodies to Chk1 (anti-Chk1) and the phosphorylated Ser296 (α-p296) and Ser317 (α-p317) sites in Chk1.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: (A) Phosphorylation of Chk1 in HeLa cell extracts. HeLa cytoplasmic extract (S100), nuclear extract (HNE) and a mixture of equal volumes of the two (HNE/S100) were incubated at 30 °C for 30 min with or without an ATP-regenerating system, 10 μM OA and 50 ng/μl poly(dA/dT)70, as indicated. One sample contained 10 mM EDTA to block kinase activity. Western blots of protein separated by gel electrophoresis were probed with antibodies to Chk1 (anti-Chk1) or the phosphorylated Ser345 site in Chk1 (α-p345). (B) Time course of Chk1 phosphorylation. The extract was incubated with OA and poly(dA/dT)70 for the times shown and blotted with antibodies to Chk1 (anti-Chk1). (C) Chk1 is phosphorylated on Ser345 in response to dsDNA, not single-stranded DNA. The extract was incubated with OA and poly(dA/dT)70, poly(dA)70, poly(dT)70 or poly(dA/dT)40 as indicated, then Western blotted with either anti-Chk1 or α-p345. (D) Phosphorylation of Chk1 on Ser296 and Ser317 in response to oligonucleotides. The extract was incubated with OA and poly(dA/dT)70 as indicated and Western blotted with antibodies to Chk1 (anti-Chk1) and the phosphorylated Ser296 (α-p296) and Ser317 (α-p317) sites in Chk1.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Incubation, Blocking Assay, Activity Assay, Western Blot, Nucleic Acid Electrophoresis

(A) Phosphorylation of endogenous Claspin. The extract was incubated with OA and poly(dA/dT)70 as indicated and blotted with antibodies to Chk1 (anti-Chk1), the phosphorylated Ser345 site in Chk1 (α-p345) and Claspin (anti-Claspin). (B) Phosphorylation of recombinant Claspin within residues 679–1332. The extract was incubated with radiolabelled fulllength Claspin, Claspin1–678 or Claspin679–1332, with or without OA and poly(dA/dT)70 as indicated. Proteins were detected by autoradiography. (C) Dephosphorylation of recombinant Claspin. The extract was incubated with radiolabelled His6-tagged full-length Claspin or Claspin679–1332, with or without OA+poly(dA/dT)70, as indicated. Proteins were precipitated, incubated further with or without protein phosphatase (λ-PPase), separated by SDS/PAGE and detected by autoradiography.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: (A) Phosphorylation of endogenous Claspin. The extract was incubated with OA and poly(dA/dT)70 as indicated and blotted with antibodies to Chk1 (anti-Chk1), the phosphorylated Ser345 site in Chk1 (α-p345) and Claspin (anti-Claspin). (B) Phosphorylation of recombinant Claspin within residues 679–1332. The extract was incubated with radiolabelled fulllength Claspin, Claspin1–678 or Claspin679–1332, with or without OA and poly(dA/dT)70 as indicated. Proteins were detected by autoradiography. (C) Dephosphorylation of recombinant Claspin. The extract was incubated with radiolabelled His6-tagged full-length Claspin or Claspin679–1332, with or without OA+poly(dA/dT)70, as indicated. Proteins were precipitated, incubated further with or without protein phosphatase (λ-PPase), separated by SDS/PAGE and detected by autoradiography.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Incubation, Recombinant, Autoradiography, De-Phosphorylation Assay, SDS Page

The extract was incubated with radiolabelled full-length Claspin (1–1332), Claspin679–1332 or Claspin1–678, with or without OA and poly(dA/dT)70 as indicated, then precipitated (IP) with Protein G Dynal beads bound to either anti-Chk1 or control antibodies (both sheep). Beads were washed and proteins were boiled off the beads and analysed by autoradiography or blotting for Chk1 using a mouse monoclonal antibody. Extracts before precipitation were also analysed.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: The extract was incubated with radiolabelled full-length Claspin (1–1332), Claspin679–1332 or Claspin1–678, with or without OA and poly(dA/dT)70 as indicated, then precipitated (IP) with Protein G Dynal beads bound to either anti-Chk1 or control antibodies (both sheep). Beads were washed and proteins were boiled off the beads and analysed by autoradiography or blotting for Chk1 using a mouse monoclonal antibody. Extracts before precipitation were also analysed.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Incubation, Autoradiography

(A) Alignment of putative Chk1-binding domains in Claspin homologues from different vertebrates. Regions where the sequence is conserved between three species (including either serine or threonine residues) are shaded. The position of aligned putative Ser-Gly or Thr-Gly phosphorylation sites is indicated. (B) Mutational analysis of the putative Chk1-binding domain in human Claspin. Claspin679–1332 (WT, upper panel) and mutants in which each of the three putative phosphorylation sites, Thr916, Ser946 and Ser982, were changed to alanine (T916A, S945A and S982A) as single (middle panel) or double (lower panel) mutants were incubated with extract, OA and poly(dA/dT)70. Immunoprecipitations with anti-Chk1 or control sheep antibodies were performed and bound proteins were analysed by blotting for Chk1 with a mouse monoclonal antibody (anti-Chk1) or by autoradiography for Claspin679–1332.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: (A) Alignment of putative Chk1-binding domains in Claspin homologues from different vertebrates. Regions where the sequence is conserved between three species (including either serine or threonine residues) are shaded. The position of aligned putative Ser-Gly or Thr-Gly phosphorylation sites is indicated. (B) Mutational analysis of the putative Chk1-binding domain in human Claspin. Claspin679–1332 (WT, upper panel) and mutants in which each of the three putative phosphorylation sites, Thr916, Ser946 and Ser982, were changed to alanine (T916A, S945A and S982A) as single (middle panel) or double (lower panel) mutants were incubated with extract, OA and poly(dA/dT)70. Immunoprecipitations with anti-Chk1 or control sheep antibodies were performed and bound proteins were analysed by blotting for Chk1 with a mouse monoclonal antibody (anti-Chk1) or by autoradiography for Claspin679–1332.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Binding Assay, Sequencing, Incubation, Autoradiography

(A) Chk1-binding motif consensus peptide. Human (residues 908–923 and 937–952) and Xenopus (residues 856–871 and 887–102) repeat Chk1-binding motifs are aligned with a synthetic peptide consensus sequence. The serine residue replaced by an alanine or phosphorylated is indicated by an asterisk. (B) Disruption of the interaction between Claspin and Chk1. The extract was incubated with [35S]Claspin679–1332, OA and poly(dA/dT)70, with further additions of buffer or 1 μg/μl phosphoserine peptide (PS), alanine peptide (AS), serine peptide (SS) or an unrelated control peptide. Proteins precipitating with sheep anti-Chk1 or control antibodies were analysed by immunoblotting with mouse anti-Chk1 or autoradiography. (C) Inhibition of Chk1 phosphorylation. The extract was incubated with OA, poly(dA/dT)70 and different concentrations of PS (top panels), AS (middle panels) or SS (bottom panels). Samples were analysed by Western blotting with anti-Chk1 and α-p345 antibodies.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: (A) Chk1-binding motif consensus peptide. Human (residues 908–923 and 937–952) and Xenopus (residues 856–871 and 887–102) repeat Chk1-binding motifs are aligned with a synthetic peptide consensus sequence. The serine residue replaced by an alanine or phosphorylated is indicated by an asterisk. (B) Disruption of the interaction between Claspin and Chk1. The extract was incubated with [35S]Claspin679–1332, OA and poly(dA/dT)70, with further additions of buffer or 1 μg/μl phosphoserine peptide (PS), alanine peptide (AS), serine peptide (SS) or an unrelated control peptide. Proteins precipitating with sheep anti-Chk1 or control antibodies were analysed by immunoblotting with mouse anti-Chk1 or autoradiography. (C) Inhibition of Chk1 phosphorylation. The extract was incubated with OA, poly(dA/dT)70 and different concentrations of PS (top panels), AS (middle panels) or SS (bottom panels). Samples were analysed by Western blotting with anti-Chk1 and α-p345 antibodies.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Binding Assay, Sequencing, Incubation, Western Blot, Autoradiography, Inhibition

The extract was incubated with radiolabelled Claspin679–1332, radiolabelled GST-CKBD, OA+poly(dA/dT)70 and the following additions: staurosporine (staur), UCN01, roscovitine (rosco) and caffeine (upper panels); Gö6976, Ro318220, Bim1, SB203580, rapamycin (Rapa), PD98059 and H89 (lower panels). Proteins were detected by immunoblotting with antibodies raised against Chk1 (anti-Chk1) or specific phosphorylation sites on Chk1 (α-p345, α-p317 and α-p296). Claspin679–1332 and GST–CKBD proteins were detected by autoradiography.

Journal:

Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

doi: 10.1042/BJ20041966

Figure Lengend Snippet: The extract was incubated with radiolabelled Claspin679–1332, radiolabelled GST-CKBD, OA+poly(dA/dT)70 and the following additions: staurosporine (staur), UCN01, roscovitine (rosco) and caffeine (upper panels); Gö6976, Ro318220, Bim1, SB203580, rapamycin (Rapa), PD98059 and H89 (lower panels). Proteins were detected by immunoblotting with antibodies raised against Chk1 (anti-Chk1) or specific phosphorylation sites on Chk1 (α-p345, α-p317 and α-p296). Claspin679–1332 and GST–CKBD proteins were detected by autoradiography.

Article Snippet: Commercial antibodies used were as follows: rabbit anti-Claspin (Ab73; Bethyl Laboratories, Montgomery, TX, U.S.A.); goat anti-ATR [FRP1 (FRAP-related protein1); N-19; Santa Cruz Biotechnology, Autogen Bioclear, calne, Wiltshire, U.K.]; mouse monoclonal anti-Chk1 (G4; Santa Cruz Biotechnology) for Western blotting; rabbit anti-Chk1 phosphorylated Ser 345 , Ser 317 and Ser 296 sites (Cell Signaling Technology, Beverly, MA, U.S.A.); and mouse monoclonal anti-tetra-His (Qiagen, Crawley, West Sussex, U.K.).

Techniques: Incubation, Western Blot, Autoradiography