rat anti orc1 Search Results


orc1  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc orc1
( A ) Strategy for insertion of a blasticidin gene and poly A site in the fourth exon of ORC2 at aa 40 of ORC2. ( B ) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC2+/+. ORC2 Knockout clones, B2 and BP8 have an insert on both alleles of ORC2 as indicated by the absence of 0.6 kb PCR product. ( C ) Verification of antibodies recognizing N-terminal or C-terminal parts of ORC2. Recombinant <t>ORC2</t> <t>protein</t> halves with Flag epitope tags were expressed and blotted with indicated antibodies. Ponceau S staining of total protein shows equal loading of lanes.* indicates full length endogenous ORC2 protein. Arrow indicates recombinant protein. ( D ) Quantitative Western blot for ORC2 with an antibody recognizing the N-terminal half of ORC2. Indicated amount of lysate loaded in each lane. ( E ) Western blot with antibody recognizing C-terminal half of ORC2. * Non specific band ( F ) Input cell lysate and immunoprecipitates of ORC2 immunoblotted for ORC2. Darker exposure of the top blots is shown in the middle. HSP90 in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. ( G ) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC2 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. ( H ) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. Ponceau S staining of histones serves as loading control for chromatin fractions. For each panel, all the lanes are from the same blot and exposure. ( I ) Comparison of Coomassie Brilliant Blue signal of pure BSA and recombinant purified GST-ORC2 to show that the top-most band in the ORC2 lane is at 170 ng/ 10 μl. ( J ) Immunoblot with different amounts of cell lysate with the GST-ORC2 to show that 1×10e5 cells give an ORC2 signal equal to 2.54 ng (1.4 fold of 1.67 ng) of GST-ORC2, which corresponds to 153×10e8 molecules of GST-ORC2. ( K ) Western blot of ORC2 in HBEC and 293T cell lines. Ponceau S staining of total protein or immunoblot of Chk1 show equal loading of the pairs of lanes. DOI: http://dx.doi.org/10.7554/eLife.19084.003
Orc1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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santa cruz anti orc1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology santa cruz anti orc1
Antibodies Used
Santa Cruz Anti Orc1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+orc1/rat+brain+extract/pmc01606514-116-234-234
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rat anti orc1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology rat anti orc1
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Rat Anti Orc1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+orc1/ORC1+Antibody/pmc00275432-370-13-24
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anti rat cy 3  (Jackson Immuno)
96
Jackson Immuno anti rat cy 3
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Anti Rat Cy 3, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-orc3  (US Biological Life Sciences)
90
US Biological Life Sciences anti-orc3
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Anti Orc3, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+orc1/anti+orc3/pm16762929-74-10-11
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anti orc2  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti orc2
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Anti Orc2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+orc1/ORC2+Antibody/10__1074_slash_jbc__m603873200-74-6-7
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n a rabbit anti orc1 méndez  (Proteintech)
92
Proteintech n a rabbit anti orc1 méndez
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
N A Rabbit Anti Orc1 Méndez, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+orc1/SLC25A15+Antibody/pm38581679-260-55-89
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orc2  (Cell Signaling Technology Inc)
92
Cell Signaling Technology Inc orc2
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Orc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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chk2  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc chk2
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Chk2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gapdh  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc gapdh
Fig. 1. Persistence of <t>ORC1</t> into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).
Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cdk2  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc cdk2
Co-treatment of SHR2554 and HBI8000 induces apoptosis, cell cycle arrest in the G1/S phase and change of histone modification. ( a , b ) Combination treatment induced G1 phase arrest in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. “–” indicated no inhibitor treatment. Then cell cycle was assessed by flow cytometry and cell-cycle-related proteins <t>(CDK2,</t> CDK4, CDK6, P21) were detected by Western blot. ( c , d ) Combination treatment prompted apoptosis in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 72 h. Then apoptosis determined by FITC+ PI− cells and FITC+ PI+ cells was assessed by flow cytometry and apoptosis-related proteins (Cleaved PARP, Caspase 3, XIAP, Mcl-1, Bcl-xL) were detected by Western blot. ( e ) Combination treatment increased acetylation of H3K27 in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. Then cells were collected and H3K27me3, H3K27ac were detected by Western blot. Representative figures are presented. Data are expressed as mean ± SD of three independent experiments and representative figures are presented. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with SHR2554 group. Detailed information about Western Blot can be found at .
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Image Search Results


( A ) Strategy for insertion of a blasticidin gene and poly A site in the fourth exon of ORC2 at aa 40 of ORC2. ( B ) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC2+/+. ORC2 Knockout clones, B2 and BP8 have an insert on both alleles of ORC2 as indicated by the absence of 0.6 kb PCR product. ( C ) Verification of antibodies recognizing N-terminal or C-terminal parts of ORC2. Recombinant ORC2 protein halves with Flag epitope tags were expressed and blotted with indicated antibodies. Ponceau S staining of total protein shows equal loading of lanes.* indicates full length endogenous ORC2 protein. Arrow indicates recombinant protein. ( D ) Quantitative Western blot for ORC2 with an antibody recognizing the N-terminal half of ORC2. Indicated amount of lysate loaded in each lane. ( E ) Western blot with antibody recognizing C-terminal half of ORC2. * Non specific band ( F ) Input cell lysate and immunoprecipitates of ORC2 immunoblotted for ORC2. Darker exposure of the top blots is shown in the middle. HSP90 in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. ( G ) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC2 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. ( H ) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. Ponceau S staining of histones serves as loading control for chromatin fractions. For each panel, all the lanes are from the same blot and exposure. ( I ) Comparison of Coomassie Brilliant Blue signal of pure BSA and recombinant purified GST-ORC2 to show that the top-most band in the ORC2 lane is at 170 ng/ 10 μl. ( J ) Immunoblot with different amounts of cell lysate with the GST-ORC2 to show that 1×10e5 cells give an ORC2 signal equal to 2.54 ng (1.4 fold of 1.67 ng) of GST-ORC2, which corresponds to 153×10e8 molecules of GST-ORC2. ( K ) Western blot of ORC2 in HBEC and 293T cell lines. Ponceau S staining of total protein or immunoblot of Chk1 show equal loading of the pairs of lanes. DOI: http://dx.doi.org/10.7554/eLife.19084.003

Journal: eLife

Article Title: Two subunits of human ORC are dispensable for DNA replication and proliferation

doi: 10.7554/eLife.19084

Figure Lengend Snippet: ( A ) Strategy for insertion of a blasticidin gene and poly A site in the fourth exon of ORC2 at aa 40 of ORC2. ( B ) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC2+/+. ORC2 Knockout clones, B2 and BP8 have an insert on both alleles of ORC2 as indicated by the absence of 0.6 kb PCR product. ( C ) Verification of antibodies recognizing N-terminal or C-terminal parts of ORC2. Recombinant ORC2 protein halves with Flag epitope tags were expressed and blotted with indicated antibodies. Ponceau S staining of total protein shows equal loading of lanes.* indicates full length endogenous ORC2 protein. Arrow indicates recombinant protein. ( D ) Quantitative Western blot for ORC2 with an antibody recognizing the N-terminal half of ORC2. Indicated amount of lysate loaded in each lane. ( E ) Western blot with antibody recognizing C-terminal half of ORC2. * Non specific band ( F ) Input cell lysate and immunoprecipitates of ORC2 immunoblotted for ORC2. Darker exposure of the top blots is shown in the middle. HSP90 in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. ( G ) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC2 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. ( H ) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. Ponceau S staining of histones serves as loading control for chromatin fractions. For each panel, all the lanes are from the same blot and exposure. ( I ) Comparison of Coomassie Brilliant Blue signal of pure BSA and recombinant purified GST-ORC2 to show that the top-most band in the ORC2 lane is at 170 ng/ 10 μl. ( J ) Immunoblot with different amounts of cell lysate with the GST-ORC2 to show that 1×10e5 cells give an ORC2 signal equal to 2.54 ng (1.4 fold of 1.67 ng) of GST-ORC2, which corresponds to 153×10e8 molecules of GST-ORC2. ( K ) Western blot of ORC2 in HBEC and 293T cell lines. Ponceau S staining of total protein or immunoblot of Chk1 show equal loading of the pairs of lanes. DOI: http://dx.doi.org/10.7554/eLife.19084.003

Article Snippet: ORC2 ( ) , ORC2N ( ) (sc-32734, Santa Cruz Biotechnology, Dallas, TX, RRID: AB_2157726 ), ORC2C ( ) (sc-13238, Santa Cruz Biotechnology, RRID: AB_2157715 ), MCM3 (sc-9850, Santa Cruz Biotechnology, RRID: AB_2142269 ), HSP90 (sc-13119, Santa Cruz Biotechnology, RRID: AB_675659 ), Cdt1 , ORC3, ORC4, ORC5, and ORC6 , ORC1 (4731, Cell Signaling Technology, Danvers, MA, RRID: AB_2157583 ), CDC6 (3387, Cell Signaling Technology, RRID: AB_2078525 ), p-Chk1 (2341, Cell Signaling Technology, RRID: AB_330023 ), p-Chk2 (2661, Cell Signaling Technology, RRID: AB_331479 ), Chk2 (3440, Cell Signaling Technology, RRID: AB_2229490 ), p-H2AX (2577, Cell Signaling Technology, RRID: AB_2118011 ), and H2AX (2595, Cell Signaling Technology, RRID: AB_10694556 ), MCM5 (A300-195A, Bethyl Laboratories, Inc Montgomery, TX, RRID: AB_185552 ), MCM7 (A300-128A, Bethyl Laboratories, Inc., RRID: AB_2142821 ), FLAG (F1804, Sigma, RRID: AB_262044 ), and Chk1 (NB100-464, Novus Biologicals, LLC, Littleton, CO, RRID: AB_10002158 ).

Techniques: Clone Assay, Knock-Out, Recombinant, FLAG-tag, Staining, Western Blot, Control, Comparison, Purification

( A ) Strategy for insertion of a blasticidin gene and poly A site after first methionine of ORC1 in the second exon. ( B ) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC1+/+. ORC1 Knockout clones, B14, B48 and BP32 have an insert on both alleles of ORC1 as indicated by the absence of 0.6 kb PCR product. ( C ) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC1 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. ( D ) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. For each panel, all the lanes are from the same blot and exposure. ( E ) Input cell lysate and immunoprecipitates of ORC1 immunoblotted for ORC1. Darker exposure of the top blots is shown in the middle. Tubulin in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. DOI: http://dx.doi.org/10.7554/eLife.19084.005

Journal: eLife

Article Title: Two subunits of human ORC are dispensable for DNA replication and proliferation

doi: 10.7554/eLife.19084

Figure Lengend Snippet: ( A ) Strategy for insertion of a blasticidin gene and poly A site after first methionine of ORC1 in the second exon. ( B ) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC1+/+. ORC1 Knockout clones, B14, B48 and BP32 have an insert on both alleles of ORC1 as indicated by the absence of 0.6 kb PCR product. ( C ) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC1 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. ( D ) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. For each panel, all the lanes are from the same blot and exposure. ( E ) Input cell lysate and immunoprecipitates of ORC1 immunoblotted for ORC1. Darker exposure of the top blots is shown in the middle. Tubulin in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. DOI: http://dx.doi.org/10.7554/eLife.19084.005

Article Snippet: ORC2 ( ) , ORC2N ( ) (sc-32734, Santa Cruz Biotechnology, Dallas, TX, RRID: AB_2157726 ), ORC2C ( ) (sc-13238, Santa Cruz Biotechnology, RRID: AB_2157715 ), MCM3 (sc-9850, Santa Cruz Biotechnology, RRID: AB_2142269 ), HSP90 (sc-13119, Santa Cruz Biotechnology, RRID: AB_675659 ), Cdt1 , ORC3, ORC4, ORC5, and ORC6 , ORC1 (4731, Cell Signaling Technology, Danvers, MA, RRID: AB_2157583 ), CDC6 (3387, Cell Signaling Technology, RRID: AB_2078525 ), p-Chk1 (2341, Cell Signaling Technology, RRID: AB_330023 ), p-Chk2 (2661, Cell Signaling Technology, RRID: AB_331479 ), Chk2 (3440, Cell Signaling Technology, RRID: AB_2229490 ), p-H2AX (2577, Cell Signaling Technology, RRID: AB_2118011 ), and H2AX (2595, Cell Signaling Technology, RRID: AB_10694556 ), MCM5 (A300-195A, Bethyl Laboratories, Inc Montgomery, TX, RRID: AB_185552 ), MCM7 (A300-128A, Bethyl Laboratories, Inc., RRID: AB_2142821 ), FLAG (F1804, Sigma, RRID: AB_262044 ), and Chk1 (NB100-464, Novus Biologicals, LLC, Littleton, CO, RRID: AB_10002158 ).

Techniques: Clone Assay, Knock-Out, Western Blot, Control

( A ) Growth curves of indicated clones of cells over four days, expressed as MTT absorbance relative to the level at day 1. (Mean ± S.D.; n = 4 biological replicates) Cells after passage for 1 month or six months. ( B ) FACS profile of propidium-iodide stained cell-cycle asynchronous cells from indicated clones. ( C ) Cells arrested in double-thymidine block released into nocodazole containing medium and harvested at indicated times after release to measure rate of progression through S phase. AS: asynchronous cells. The red dotted lines indicate cells with G1 and G2 DNA content. ( D ) Molecular combing of chromosomal DNA after a pulse of CldU for 30 min chased with a pulse of IdU for 30 min. Box and whiskers plot for fork progression rate and inter-origin distance of indicated clones of cells. (P value < 4.6e-06, two-sided Wilcoxon rank sum test for two samples N = number of tracks counted) (Inter origin disntance N = 91(WT), 131(ORC1B14), 174(ORC1BP32) p: Statistical significance of any difference between WT and ORC1-/- cells. ( E ) Overlap of the BrIP-seq peaks between WT and ORC1-/- cells. ( F ) Box and whiskers plot for inter-origin distances (measured by BrIP-seq) for each chromosome in WT and ORC1-/- cells. The median inter-origin distance for all chromosomes together indicated at bottom right. ( G ) Circos plot of Origins mapped by BrIP-seq for chromosome 1. Outer circle: the chromosome with the karytotyping bands. Inner two circles: the locations of BrIPseq peaks in the WT and ORC1-/- cell lines. ( H ) Distribution of BrIP-seq mapped origins in ORC1-/- cells relative to distance from Transcription Start Sites (TSS). DOI: http://dx.doi.org/10.7554/eLife.19084.006

Journal: eLife

Article Title: Two subunits of human ORC are dispensable for DNA replication and proliferation

doi: 10.7554/eLife.19084

Figure Lengend Snippet: ( A ) Growth curves of indicated clones of cells over four days, expressed as MTT absorbance relative to the level at day 1. (Mean ± S.D.; n = 4 biological replicates) Cells after passage for 1 month or six months. ( B ) FACS profile of propidium-iodide stained cell-cycle asynchronous cells from indicated clones. ( C ) Cells arrested in double-thymidine block released into nocodazole containing medium and harvested at indicated times after release to measure rate of progression through S phase. AS: asynchronous cells. The red dotted lines indicate cells with G1 and G2 DNA content. ( D ) Molecular combing of chromosomal DNA after a pulse of CldU for 30 min chased with a pulse of IdU for 30 min. Box and whiskers plot for fork progression rate and inter-origin distance of indicated clones of cells. (P value < 4.6e-06, two-sided Wilcoxon rank sum test for two samples N = number of tracks counted) (Inter origin disntance N = 91(WT), 131(ORC1B14), 174(ORC1BP32) p: Statistical significance of any difference between WT and ORC1-/- cells. ( E ) Overlap of the BrIP-seq peaks between WT and ORC1-/- cells. ( F ) Box and whiskers plot for inter-origin distances (measured by BrIP-seq) for each chromosome in WT and ORC1-/- cells. The median inter-origin distance for all chromosomes together indicated at bottom right. ( G ) Circos plot of Origins mapped by BrIP-seq for chromosome 1. Outer circle: the chromosome with the karytotyping bands. Inner two circles: the locations of BrIPseq peaks in the WT and ORC1-/- cell lines. ( H ) Distribution of BrIP-seq mapped origins in ORC1-/- cells relative to distance from Transcription Start Sites (TSS). DOI: http://dx.doi.org/10.7554/eLife.19084.006

Article Snippet: ORC2 ( ) , ORC2N ( ) (sc-32734, Santa Cruz Biotechnology, Dallas, TX, RRID: AB_2157726 ), ORC2C ( ) (sc-13238, Santa Cruz Biotechnology, RRID: AB_2157715 ), MCM3 (sc-9850, Santa Cruz Biotechnology, RRID: AB_2142269 ), HSP90 (sc-13119, Santa Cruz Biotechnology, RRID: AB_675659 ), Cdt1 , ORC3, ORC4, ORC5, and ORC6 , ORC1 (4731, Cell Signaling Technology, Danvers, MA, RRID: AB_2157583 ), CDC6 (3387, Cell Signaling Technology, RRID: AB_2078525 ), p-Chk1 (2341, Cell Signaling Technology, RRID: AB_330023 ), p-Chk2 (2661, Cell Signaling Technology, RRID: AB_331479 ), Chk2 (3440, Cell Signaling Technology, RRID: AB_2229490 ), p-H2AX (2577, Cell Signaling Technology, RRID: AB_2118011 ), and H2AX (2595, Cell Signaling Technology, RRID: AB_10694556 ), MCM5 (A300-195A, Bethyl Laboratories, Inc Montgomery, TX, RRID: AB_185552 ), MCM7 (A300-128A, Bethyl Laboratories, Inc., RRID: AB_2142821 ), FLAG (F1804, Sigma, RRID: AB_262044 ), and Chk1 (NB100-464, Novus Biologicals, LLC, Littleton, CO, RRID: AB_10002158 ).

Techniques: Clone Assay, Staining, Blocking Assay

Antibodies Used

Journal:

Article Title: Cell-Cycle Markers in a Transgenic Mouse Model of Human Tauopathy

doi: 10.2353/ajpath.2006.050540

Figure Lengend Snippet: Antibodies Used

Article Snippet: They included antibodies to cyclins (A, B, D1, and E), CDKs 1 and 2, Cdc25A, Cdc25C, Cks1, Myt1/Wee1, Polo kinase, CDK inhibitors (p16Ink4a, p18Ink4c, p19ARF, p21Cip1, and p27Kip1), ORC1, Aurora2, MPM2, phospho-histone H3, mitotic cells, and the retinoblastoma (Rb) protein. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antibodies Dilutions used for immunohistochemistry Dilutions used for immunoblots Source Anti-Tau BR134 1/500 1/5000 M. Goedert AT8 1/500 1/1000 Innogenetics AT100 1/500 1/1000 Innogenetics Phospho Tau (Ser422) 1/500 1/1000 Biosource Anti-CDKs Cdk2 1/100 1/200 H-298/Santa Cruz (Santa Cruz, CA) Phospho-Cdk2 (Thr160) 1/50 1/100 Santa Cruz Cdk1 (Cdc2) 1/250 1/1000 Upstate Biotechnology (Dundee, UK) Phospho-Cdk1 (Thr161) 1/200 1/1000 Cell Signaling (Danvers, MA) Anti-Cyclins Cyclin A 1/100 1/200 H-432/Santa Cruz 1/100 1/200 ab7956/Abcam (Cambridge, UK) Cyclin B 1/100 1/200 H-433/Santa Cruz 1/250 1/2000 Cell Signaling Cyclin D1 1/70 1/200 H-295/Santa Cruz Cyclin E 1/70 1/200 M20/Santa Cruz Anti-Cdk activators and inhibitors Cdc25 A 1/50 1/200 M-191/Santa Cruz Cdc25 C 1/50 1/200 TC-15/Upstate Biotechnology Phospho-Cdc25 C (Thr48) 1/100 1/1000 Cell Signaling CKs1 p9 1/50 n.d. FL-79/Santa Cruz Myt1/Wee1 1/200 1/1000 Cell Signaling Polo kinase (Plk) 1/200 1/1000 NT/Upstate Biotechnology p16 Ink4 1/50 n.d. M-156/Santa Cruz p18 Ink4 1/50 n.d. N-20/Santa Cruz p19 ARF 1/50 n.d. M-20/Santa Cruz p21 Cip1 1/50 1/200 F-5/Santa Cruz 1/50 1/200 Waf1 (Ab4)/Oncogene Research Products, (San Diego, CA) p27 Kip1 1/100 1/1000 Cell Signaling 1/50 1/200 DCS-72/Sigma-Aldrich (Poole, UK) Other markers Anti-ATM 1/70 1/200 Sc-23922/Santa Cruz Anti-ORC1 1/50 n.d. 7F6/1/Neomarkers (Fremont, CA) Anti-Aurora 2 1/100 n.d.

Techniques: Immunohistochemistry, Western Blot

Cell Cycle Markers in Human P301S Tau-Transgenic (Tg) Mice and Age-Matched Controls

Journal:

Article Title: Cell-Cycle Markers in a Transgenic Mouse Model of Human Tauopathy

doi: 10.2353/ajpath.2006.050540

Figure Lengend Snippet: Cell Cycle Markers in Human P301S Tau-Transgenic (Tg) Mice and Age-Matched Controls

Article Snippet: They included antibodies to cyclins (A, B, D1, and E), CDKs 1 and 2, Cdc25A, Cdc25C, Cks1, Myt1/Wee1, Polo kinase, CDK inhibitors (p16Ink4a, p18Ink4c, p19ARF, p21Cip1, and p27Kip1), ORC1, Aurora2, MPM2, phospho-histone H3, mitotic cells, and the retinoblastoma (Rb) protein. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antibodies Dilutions used for immunohistochemistry Dilutions used for immunoblots Source Anti-Tau BR134 1/500 1/5000 M. Goedert AT8 1/500 1/1000 Innogenetics AT100 1/500 1/1000 Innogenetics Phospho Tau (Ser422) 1/500 1/1000 Biosource Anti-CDKs Cdk2 1/100 1/200 H-298/Santa Cruz (Santa Cruz, CA) Phospho-Cdk2 (Thr160) 1/50 1/100 Santa Cruz Cdk1 (Cdc2) 1/250 1/1000 Upstate Biotechnology (Dundee, UK) Phospho-Cdk1 (Thr161) 1/200 1/1000 Cell Signaling (Danvers, MA) Anti-Cyclins Cyclin A 1/100 1/200 H-432/Santa Cruz 1/100 1/200 ab7956/Abcam (Cambridge, UK) Cyclin B 1/100 1/200 H-433/Santa Cruz 1/250 1/2000 Cell Signaling Cyclin D1 1/70 1/200 H-295/Santa Cruz Cyclin E 1/70 1/200 M20/Santa Cruz Anti-Cdk activators and inhibitors Cdc25 A 1/50 1/200 M-191/Santa Cruz Cdc25 C 1/50 1/200 TC-15/Upstate Biotechnology Phospho-Cdc25 C (Thr48) 1/100 1/1000 Cell Signaling CKs1 p9 1/50 n.d. FL-79/Santa Cruz Myt1/Wee1 1/200 1/1000 Cell Signaling Polo kinase (Plk) 1/200 1/1000 NT/Upstate Biotechnology p16 Ink4 1/50 n.d. M-156/Santa Cruz p18 Ink4 1/50 n.d. N-20/Santa Cruz p19 ARF 1/50 n.d. M-20/Santa Cruz p21 Cip1 1/50 1/200 F-5/Santa Cruz 1/50 1/200 Waf1 (Ab4)/Oncogene Research Products, (San Diego, CA) p27 Kip1 1/100 1/1000 Cell Signaling 1/50 1/200 DCS-72/Sigma-Aldrich (Poole, UK) Other markers Anti-ATM 1/70 1/200 Sc-23922/Santa Cruz Anti-ORC1 1/50 n.d. 7F6/1/Neomarkers (Fremont, CA) Anti-Aurora 2 1/100 n.d.

Techniques: Immunofluorescence, Expressing, Western Blot

Fig. 1. Persistence of ORC1 into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 1. Persistence of ORC1 into M phase. (A) Schematic of the synchronous cell cycle transition in the eye imaginal disc. As the morphogenetic furrow (MF, hereinafter marked with an arrowhead) sweeps from posterior (P) to anterior (A), most cells undergo a synchronous transition and then enter a prolonged G1/G0 phase. (Note that some cells behind the MF are in a prolonged G2 arrest, as shown in Figure 3.) Ahead of the furrow and in the attached antennal disc, cells cycle asynchronously. (B–K) Confocal images of eye imaginal discs near the MF (arrowhead). Endogenous ORC1 (B–F) and ORC1–GFP expressed under ORC1 transcriptional control (G–K), and CycB or PH3, as indicated. Interphase nuclei are visible in optical sections through the middle of the disc (B, D, G and I), whereas mitotic nuclei are visible in apical optical sections (C, E, F, H, J and K). Arrows in (H) are examples of late telophase nuclei (see also Figure 3K–M).

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques:

Fig. 2. Absence of ORC1 in G1 phase. FACS analysis of dissociated imaginal disc cells from transgenic animals expressing ORC1–GFP under ORC1 transcriptional control. The proportion of G1 cells in eye antennal discs is higher than in wing discs, due to the contribution of terminally differentiating cells behind the morphogenetic furrow.

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 2. Absence of ORC1 in G1 phase. FACS analysis of dissociated imaginal disc cells from transgenic animals expressing ORC1–GFP under ORC1 transcriptional control. The proportion of G1 cells in eye antennal discs is higher than in wing discs, due to the contribution of terminally differentiating cells behind the morphogenetic furrow.

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques: Transgenic Assay, Expressing

Fig. 3. Regulated proteolysis generates a normal temporal distribution of ORC1 protein even upon constitutive ORC1 transcription. Expression of various proteins under control of the GMR promoter (A–C, E–H, K and L), which is active in all cells posterior to the MF (arrowhead) in the eye disc, or the engrailed (en) promoter (I and J), which is active in all cells of the posterior compartment of the wing disc. High magnification views of the area of the eye disc boxed in (A) reveal that cells with high levels of ORC1–GFP are CycB positive with no BrdU incorporation (not shown), and therefore in G2 (B–D). FACS analysis reveals that ORC1–GFP is depleted in G1 phase eye (G and H) and wing (I and J) imaginal disc cells, even if transcription of ORC1–GFP is driven constitutively. High magnification views of cells immediately posterior to the MF reveal co-localization of ORC1–GFP and CycB following nuclear envelope breakdown and the presence of late telophase cells with paired nuclei bearing high levels of ORC1–GFP but no significant CycB (K–M).

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 3. Regulated proteolysis generates a normal temporal distribution of ORC1 protein even upon constitutive ORC1 transcription. Expression of various proteins under control of the GMR promoter (A–C, E–H, K and L), which is active in all cells posterior to the MF (arrowhead) in the eye disc, or the engrailed (en) promoter (I and J), which is active in all cells of the posterior compartment of the wing disc. High magnification views of the area of the eye disc boxed in (A) reveal that cells with high levels of ORC1–GFP are CycB positive with no BrdU incorporation (not shown), and therefore in G2 (B–D). FACS analysis reveals that ORC1–GFP is depleted in G1 phase eye (G and H) and wing (I and J) imaginal disc cells, even if transcription of ORC1–GFP is driven constitutively. High magnification views of cells immediately posterior to the MF reveal co-localization of ORC1–GFP and CycB following nuclear envelope breakdown and the presence of late telophase cells with paired nuclei bearing high levels of ORC1–GFP but no significant CycB (K–M).

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques: Expressing, BrdU Incorporation Assay

Fig. 4. Signals that mediate regulated proteolysis of ORC1 reside in its non-conserved N-terminal domain. (A) Schema of the deletion derivatives analyzed in (B–K). Expression of various ORC1 derivatives in the eye disc under GMR promoter control (B–E, each with a high magnification view inset), also analyzed by western blot (F), northern blot (G) and dissociation into single cells followed by FACS (H–K). The gels in (F) and (G) were re-probed with the loading controls shown at the bottom of each blot. Analysis of western blots reveals that the steady-state level of ORC1 or ORC1N is ∼25% that of ORC1C. Since these samples are homogenates of cells with low and high levels of protein, it underestimates the extent of regulation per cell (probably by a factor of ∼2, based on the cell cycle distributions of H–K).

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 4. Signals that mediate regulated proteolysis of ORC1 reside in its non-conserved N-terminal domain. (A) Schema of the deletion derivatives analyzed in (B–K). Expression of various ORC1 derivatives in the eye disc under GMR promoter control (B–E, each with a high magnification view inset), also analyzed by western blot (F), northern blot (G) and dissociation into single cells followed by FACS (H–K). The gels in (F) and (G) were re-probed with the loading controls shown at the bottom of each blot. Analysis of western blots reveals that the steady-state level of ORC1 or ORC1N is ∼25% that of ORC1C. Since these samples are homogenates of cells with low and high levels of protein, it underestimates the extent of regulation per cell (probably by a factor of ∼2, based on the cell cycle distributions of H–K).

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques: Expressing, Western Blot, Northern Blot

Fig. 5. APC-dependent degradation of ORC1 in embryonic cells. Confocal images of epithelial cells just ventral to the leading edge in stage 12–13 embryos (A–H). At this stage, most cells are already in G1 of cycle 17 and only a minority of cells are in S or G2 of cycle 16, with high levels of both ORC1 and CycB (A–C). In (D–H), transcription of various proteins (as indicated) is driven by the constitutive Actin5C promoter. The few cells with high levels of ORC1–GFP or ORC1N–GFP also contain appreciable CycB and therefore are still in G2 of cycle 16 (not shown). We used northern blots (J) to select transgenic UAS lines that direct transcription of essentially identical levels of each gene, and then analyzed the steady-state level of each ORC1–GFP derivative by western blot (I). This analysis reveals that the steady-state level of ORC1C and ORC1C–NLS is 4- to 6-fold higher than ORC1–GFP and 6- to 9-fold higher than ORC1N. As in Figure 4, this analysis underestimates the extent of regulation per cell. Transmitted light microscopy reveals accumulation of CycB (K versus L) and ORC1 (M–P) in essentially every epithelial cell of fzr mutant embryos. Note that differential accumulation in internal tissues (primarily midgut) partially obscures accumulation in epithelial cells.

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 5. APC-dependent degradation of ORC1 in embryonic cells. Confocal images of epithelial cells just ventral to the leading edge in stage 12–13 embryos (A–H). At this stage, most cells are already in G1 of cycle 17 and only a minority of cells are in S or G2 of cycle 16, with high levels of both ORC1 and CycB (A–C). In (D–H), transcription of various proteins (as indicated) is driven by the constitutive Actin5C promoter. The few cells with high levels of ORC1–GFP or ORC1N–GFP also contain appreciable CycB and therefore are still in G2 of cycle 16 (not shown). We used northern blots (J) to select transgenic UAS lines that direct transcription of essentially identical levels of each gene, and then analyzed the steady-state level of each ORC1–GFP derivative by western blot (I). This analysis reveals that the steady-state level of ORC1C and ORC1C–NLS is 4- to 6-fold higher than ORC1–GFP and 6- to 9-fold higher than ORC1N. As in Figure 4, this analysis underestimates the extent of regulation per cell. Transmitted light microscopy reveals accumulation of CycB (K versus L) and ORC1 (M–P) in essentially every epithelial cell of fzr mutant embryos. Note that differential accumulation in internal tissues (primarily midgut) partially obscures accumulation in epithelial cells.

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques: Northern Blot, Transgenic Assay, Western Blot, Light Microscopy, Mutagenesis

Fig. 6. APC-mediated degradation of ORC1–GFP in imaginal cells. Co-expression of Fzr, which stimulates APC-dependent substrate degradation, significantly destabilizes ORC1–GPF in the eye disc (B versus A), whereas co-expression of Rca1, an inhibitor of Fzr, has the opposite effect (C). No effect was seen on the stability of the unregulated C-terminal domain of ORC1 (D–F). Western blots of the same disc samples are shown in (G) and (H). The distribution of BrdU incorporated during a brief pulse reveals that misexpression of neither Fzr nor Rca1 drives cells ectopically into S phase (I–K), although we see a slight effect similar to that reported (Dong et al., 1997) in the latter case. FACS analysis (L and M) reveals that misexpression of Fzr causes a modest accumulation of G2 cells (at the expense of the G1 population), whereas misexpression of Rca1 has no effect on the profile of cells in the posterior of the eye disc where the GMR promoter is active. Note that these experiments were performed in the absence of UAS-ORC1–GFP transgenes, but that expression of ORC1–GFP derivatives alone does not alter cycling of eye disc cells (Figures 2–4).

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 6. APC-mediated degradation of ORC1–GFP in imaginal cells. Co-expression of Fzr, which stimulates APC-dependent substrate degradation, significantly destabilizes ORC1–GPF in the eye disc (B versus A), whereas co-expression of Rca1, an inhibitor of Fzr, has the opposite effect (C). No effect was seen on the stability of the unregulated C-terminal domain of ORC1 (D–F). Western blots of the same disc samples are shown in (G) and (H). The distribution of BrdU incorporated during a brief pulse reveals that misexpression of neither Fzr nor Rca1 drives cells ectopically into S phase (I–K), although we see a slight effect similar to that reported (Dong et al., 1997) in the latter case. FACS analysis (L and M) reveals that misexpression of Fzr causes a modest accumulation of G2 cells (at the expense of the G1 population), whereas misexpression of Rca1 has no effect on the profile of cells in the posterior of the eye disc where the GMR promoter is active. Note that these experiments were performed in the absence of UAS-ORC1–GFP transgenes, but that expression of ORC1–GFP derivatives alone does not alter cycling of eye disc cells (Figures 2–4).

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques: Expressing, Western Blot

Fig. 8. Model of our current understanding of the temporal distribution of ORC1 in fly, human and either budding or fission yeast cells. Drosophila Cdc6 is uncharacterized and therefore omitted.

Journal:

Article Title: Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila

doi: 10.1093/emboj/cdg573

Figure Lengend Snippet: Fig. 8. Model of our current understanding of the temporal distribution of ORC1 in fly, human and either budding or fission yeast cells. Drosophila Cdc6 is uncharacterized and therefore omitted.

Article Snippet: Primary antibodies were used at the following concentrations; 1:1000 rabbit anti-CycB; 1:100 affinity-purified rat anti-ORC1; 1:6000 rabbit anti-PH3 (Upstate); and 1:3000 mouse anti-myc (9E10, Santa Cruz).

Techniques:

Co-treatment of SHR2554 and HBI8000 induces apoptosis, cell cycle arrest in the G1/S phase and change of histone modification. ( a , b ) Combination treatment induced G1 phase arrest in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. “–” indicated no inhibitor treatment. Then cell cycle was assessed by flow cytometry and cell-cycle-related proteins (CDK2, CDK4, CDK6, P21) were detected by Western blot. ( c , d ) Combination treatment prompted apoptosis in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 72 h. Then apoptosis determined by FITC+ PI− cells and FITC+ PI+ cells was assessed by flow cytometry and apoptosis-related proteins (Cleaved PARP, Caspase 3, XIAP, Mcl-1, Bcl-xL) were detected by Western blot. ( e ) Combination treatment increased acetylation of H3K27 in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. Then cells were collected and H3K27me3, H3K27ac were detected by Western blot. Representative figures are presented. Data are expressed as mean ± SD of three independent experiments and representative figures are presented. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with SHR2554 group. Detailed information about Western Blot can be found at .

Journal: Cancers

Article Title: The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma

doi: 10.3390/cancers13174249

Figure Lengend Snippet: Co-treatment of SHR2554 and HBI8000 induces apoptosis, cell cycle arrest in the G1/S phase and change of histone modification. ( a , b ) Combination treatment induced G1 phase arrest in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. “–” indicated no inhibitor treatment. Then cell cycle was assessed by flow cytometry and cell-cycle-related proteins (CDK2, CDK4, CDK6, P21) were detected by Western blot. ( c , d ) Combination treatment prompted apoptosis in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 72 h. Then apoptosis determined by FITC+ PI− cells and FITC+ PI+ cells was assessed by flow cytometry and apoptosis-related proteins (Cleaved PARP, Caspase 3, XIAP, Mcl-1, Bcl-xL) were detected by Western blot. ( e ) Combination treatment increased acetylation of H3K27 in DLBCL cells. Cells were treated with indicated concentrations of SHR2554 and HBI8000 for 48 h. Then cells were collected and H3K27me3, H3K27ac were detected by Western blot. Representative figures are presented. Data are expressed as mean ± SD of three independent experiments and representative figures are presented. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with SHR2554 group. Detailed information about Western Blot can be found at .

Article Snippet: Antibodies against Caspase-3 (#9662S), Mcl-1 (#5453S), Bcl-xl (#2764S), XIAP (#2045S), CDK2 (#2546S), CDK4 (#12790S), CDK6(#13331S), H3K27me3(#9733T), H3K27ac(#9649S), H3(#4499S), EZH2(#5246S), P21(#2947S), PARP (#9532S, #5625S), ORC1 (#4371) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Modification, Flow Cytometry, Western Blot