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phosphorylated chk1 ser 345  (Bethyl)

 
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    Bethyl phosphorylated chk1 ser 345
    ( 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 ) <t>Chk1</t> phosphorylation was examined in wild-type and the mutant cells treated with (+) or without (-) 0.01% 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.
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    Images

    1) Product Images from "Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast"

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

    Journal: PLOS Genetics

    doi: 10.1371/journal.pgen.1010691

    ( 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.
    Figure Legend 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.

    Techniques Used: Spot Test, Incubation, Mutagenesis, Western Blot, SDS Page, Staining, Quantitation Assay

    ( 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.
    Figure Legend 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.

    Techniques Used: Quantitation Assay, Western Blot, Mobility Shift

    (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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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.
    Figure Legend 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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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.

    Techniques Used: Binding Assay, Mutagenesis, Spot Test, Mobility Shift

    ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , respectively.
    Figure Legend Snippet: ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , respectively.

    Techniques Used: Spot Test, Mutagenesis, Quantitation Assay, Mobility Shift



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    ( 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 ) <t>Chk1</t> phosphorylation was examined in wild-type and the mutant cells treated with (+) or without (-) 0.01% 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.
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    ( 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.

    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, Mutagenesis, Western Blot, SDS Page, Staining, Quantitation Assay

    ( 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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: Quantitation Assay, Western Blot, Mobility Shift

    (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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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, Mobility Shift

    ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , respectively.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    Figure Lengend Snippet: ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , 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, Mutagenesis, Quantitation Assay, Mobility Shift

    ( 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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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 rad3Δ (NR1826), cds1Δ (GBY191), and chk1Δ (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 B 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 (-) 0.01% 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.

    Article Snippet: After transfer to a nitrocellulose membrane, the membrane was blotted with the Chk1-pS345 antibody at the 1:3000 dilution for 3 h to detect the phosphorylated Chk1-Ser 345 in ChemiDoc (Bio-Rad).

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

    ( 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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: After transfer to a nitrocellulose membrane, the membrane was blotted with the Chk1-pS345 antibody at the 1:3000 dilution for 3 h to detect the phosphorylated Chk1-Ser 345 in ChemiDoc (Bio-Rad).

    Techniques: Quantitation Assay, Western Blot, Mobility Shift

    (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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    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 (Figs and ). Relative sensitivities to chronic (Figs and ) and acute HU treatment (Figs and ) 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: After transfer to a nitrocellulose membrane, the membrane was blotted with the Chk1-pS345 antibody at the 1:3000 dilution for 3 h to detect the phosphorylated Chk1-Ser 345 in ChemiDoc (Bio-Rad).

    Techniques: Binding Assay, Mutagenesis, Spot Test, Mobility Shift, Control

    ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , respectively.

    Journal: PLOS Genetics

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

    doi: 10.1371/journal.pgen.1010691

    Figure Lengend Snippet: ( A ) Six primary ssb1 mutants with more prominent 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 . Quantitation results from three independent blots are shown in , respectively. Chk1 phosphorylation in the six integrants was examined as in by phospho-specific antibody ( D) and by mobility shift assay (E ) and the quantitation results are shown in , 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 , respectively.

    Article Snippet: After transfer to a nitrocellulose membrane, the membrane was blotted with the Chk1-pS345 antibody at the 1:3000 dilution for 3 h to detect the phosphorylated Chk1-Ser 345 in ChemiDoc (Bio-Rad).

    Techniques: Spot Test, Mutagenesis, Quantitation Assay, Mobility Shift

    Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Activation Assay, Blocking Assay, Transduction

    Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol "+" indicates presence of the siR-Chk1 or siR-Chk1-S345A plasmids. The dash sign "-"means no plasmid was introduced in wild-type Chk1, depleted by siRNA. The cell cycle profiles of the indicated cell lines were measured 48 hours after the adenoviral transduction by flow cytometric analysis ( A ). Expression of endogenous or siRNA-resistant Chk1 constructs from indicated cell lines was confirmed by Western blot analysis using anti-Chk1 antibody at the same time of flow cytometric analysis ( B ). Note that the siR-Chk1 or siR-Chk1-Ser345A gene products cannot be depleted by the normal "Chk1 siRNA" used here because silent mutations were incorporated into the Chk1 genes during site-directed mutagenesis. These silent mutations will not alter the intended protein sequences, i.e., wild type Chk1 or Chk1-Ser345A.

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Transfection, Mutagenesis, Transduction, Plasmid Preparation, Expressing, Construct, Western Blot

    Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Transduction, Western Blot

    Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Transduction, Western Blot

    Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Transduction, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Staining, Transduction, Fluorescence, Microscopy, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

    Article Snippet: It should be mentioned that the effects of Vpr on Chk1-Ser 345 phosphorylation and Cdt1 or Cdc6 were shown here in two different cell types (HeLa and CEM-SS), which suggest a general effect of Vpr on these cells.

    Techniques: Cell Culture, Transduction, Staining, Western Blot

    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Journal: PLoS ONE

    Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

    doi: 10.1371/journal.pone.0099397

    Figure Lengend Snippet: (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Article Snippet: Antibodies used include ATR-N19 (Santa Cruz Biotechnology), HA (Covance), CHK1-G4 (Santa Cruz Biotechnology), FLAG-M2 (Sigma), ATRIP403 , MCM2 (BD Transduction Labs), phosphorylated Ser-317 CHK1 (Cell Signaling Technology), phosphorylated Ser-345 CHK1 (Cell Signaling Technology), and phosphorylated Ser-10 Histone H3 (Cell Signaling Technology).

    Techniques: Clone Assay, Expressing, SDS Page, Western Blot