anti phosphorylated chk1 ser 345  (Cell Signaling Technology Inc)

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

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

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

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

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