Cellular localization of endogenous RFC140 and PCNA in asynchronous 293T cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> S1, S2 and P2 indicate cytoplasmic, nucleoplasm (soluble nuclear) and chromatin/nuclear matrix (insoluble nuclear) fractions, respectively. MEK2 and ORC2 blots are represented as a markers specific to cytoplasmic and chromatin fractions, respectively

() Recruitment of EGFP-tagged proteins related to DNA repair synthesis to DNA damage induced by a 365-nm UVA-laser

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> (A) Nuclei (arrowheads) of HeLa cells expressing EGFP-RFC140, EGFP-PCNA and EGFP-Polδ1, were irradiated with a 365-nm UVA laser as described in ‘Materials and Methods’ section. () Accumulation of EGFP-fusions, RFC140 (square), PCNA (triangle) and Polδ1 (circle) in (A) was measured as the fold increase of fluorescence intensity at an irradiated site. Data were taken from five independent experiments. Error bars represent standard errors. () Intensity at laser irradiation sites of EGFP-fusions and five RFC subunits was plotted as in (B). () Maximum intensity (MI) and the time to reach MI ( MAX) were represented in each GFP-fusion. A half of MI (1/2 MI) was calculated as 0.5 × (MI − 1) + 1. Thus, 1/2 MI indicates the time to reach 50% of MI

Accumulation kinetics of a series of deletion fragments with or without F6A/F7A substitution

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> () Sequence alignment of the N-terminal portion of RFC140 orthologs from nine eukaryotes. Approximately 10 amino-acid residues are indicated. Red-colored residues are highly conserved among all species, and are indicated as the PIP-box (see text). EGFP-fused fragments 1–369 (), 1–397 (), 1–493 (), 1–733 () and full-length (1–1147, ) having normal (closed squares) or the F6A/F7A mutation (open squares) were transiently expressed in HeLa cells and accumulation was measured as the fold increase of fluorescence intensity. Error bars indicate standard error. Error bars not indicated are smaller than symbols. Pictures represent accumulation of FA mutant fragments, and are taken before and 120 min after laser irradiation. Data were taken from five independent experiments. White arrowheads indicate the laser irradiation region

Accumulation of EGFP-RFC140 and EGFP-PCNA in response to laser-induced DNA damage in xrcc1-deficient and xrcc1-proficient mouse embryonic cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> Nuclei of xrcc1-deficient (KO) or proficient (WT) mouse embryonic cells expressing EGFP-RFC140 or EGFP-PCNA were irradiated with a low dose (SSBs) or a high dose (SSBs + DSBs) of 365-nm UVA-laser light. Time-lapse pictures were taken as indicated in A

Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques