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mouse orc2 antibody  (Pacific Immunology)

 
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    Pacific Immunology mouse orc2 antibody
    ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from <t>Orc2</t> f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the <t>ORC2</t> <t>protein</t> and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.
    Mouse Orc2 Antibody, supplied by Pacific Immunology, 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/mouse+orc2+antibody/mouse+orc2+antibody/pmc12043314-226-0-16
    Average 90 stars, based on 1 article reviews
    mouse orc2 antibody - by Bioz Stars, 2026-09
    90/100 stars

    Images

    1) Product Images from "DNA replication in primary hepatocytes without the six-subunit ORC"

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    Journal: eLife

    doi: 10.7554/eLife.102915

    ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.
    Figure Legend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

    Techniques Used: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

    Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.
    Figure Legend Snippet: Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

    Techniques Used: Expressing

    ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.
    Figure Legend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

    Techniques Used: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

    ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.
    Figure Legend Snippet: ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

    Techniques Used: Two Tailed Test

    ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.
    Figure Legend Snippet: ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

    Techniques Used: Western Blot, Staining, Two Tailed Test

    ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.
    Figure Legend Snippet: ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

    Techniques Used: Staining, Knock-Out, Quantitation Assay, Two Tailed Test

    ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.
    Figure Legend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

    Techniques Used: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

    ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.
    Figure Legend Snippet: ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

    Techniques Used: Staining

    ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .
    Figure Legend Snippet: ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

    Techniques Used: Mutagenesis, Expressing

    ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.
    Figure Legend Snippet: ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

    Techniques Used: Two Tailed Test, Staining, Quantitation Assay, Imaging

    Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.
    Figure Legend Snippet: Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

    Techniques Used:

    Related Articles

    Recombinant:

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC
    Article Snippet: .. Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology). .. The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).



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    ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from <t>Orc2</t> f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the <t>ORC2</t> <t>protein</t> and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.
    Mouse Anti Orc2 Antibodies, supplied by MBL Life science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 4. Dynamic association of ORC1, RB, SUV39H1 and CDC6 proteins to the CCNE1 promoter during the cell cycle. (A–B) Nocodazole arrested U2OS cells were released for different times (3, 6 and 9 hr) and analyzed for occupancy of ORC1, RB, SUV39H1 and CDC6 proteins at the CCNE1 promoter by ChIP assay. The primer pairs used to analyze two different regions of the CCNE1 promoter are indicated. The experiments were done in triplicate with results similar to those shown. (C) Whole cell protein levels of nocodazole arrested and released U2OS cells at different time points (as indicated in hours) by immunoblotting with antibodies against ORC1, RB, SUV39H1, CDC6, Cyclin E, ORC3 and a-Tubulin. ORC, <t>Origin</t> <t>Recognition</t> <t>Complex;</t> RB, Retinoblastoma. DOI: 10.7554/eLife.12785.012
    Monoclonal Mouse Orc2 Antibody, 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
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    Image Search Results


    ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

    Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Expressing

    ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

    ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Two Tailed Test

    ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Western Blot, Staining, Two Tailed Test

    ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Staining, Knock-Out, Quantitation Assay, Two Tailed Test

    ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

    ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Staining

    ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Mutagenesis, Expressing

    ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques: Two Tailed Test, Staining, Quantitation Assay, Imaging

    Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

    Journal: eLife

    Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

    doi: 10.7554/eLife.102915

    Figure Lengend Snippet: Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

    Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

    Techniques:

    Figure 4. Dynamic association of ORC1, RB, SUV39H1 and CDC6 proteins to the CCNE1 promoter during the cell cycle. (A–B) Nocodazole arrested U2OS cells were released for different times (3, 6 and 9 hr) and analyzed for occupancy of ORC1, RB, SUV39H1 and CDC6 proteins at the CCNE1 promoter by ChIP assay. The primer pairs used to analyze two different regions of the CCNE1 promoter are indicated. The experiments were done in triplicate with results similar to those shown. (C) Whole cell protein levels of nocodazole arrested and released U2OS cells at different time points (as indicated in hours) by immunoblotting with antibodies against ORC1, RB, SUV39H1, CDC6, Cyclin E, ORC3 and a-Tubulin. ORC, Origin Recognition Complex; RB, Retinoblastoma. DOI: 10.7554/eLife.12785.012

    Journal: eLife

    Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

    doi: 10.7554/elife.12785

    Figure Lengend Snippet: Figure 4. Dynamic association of ORC1, RB, SUV39H1 and CDC6 proteins to the CCNE1 promoter during the cell cycle. (A–B) Nocodazole arrested U2OS cells were released for different times (3, 6 and 9 hr) and analyzed for occupancy of ORC1, RB, SUV39H1 and CDC6 proteins at the CCNE1 promoter by ChIP assay. The primer pairs used to analyze two different regions of the CCNE1 promoter are indicated. The experiments were done in triplicate with results similar to those shown. (C) Whole cell protein levels of nocodazole arrested and released U2OS cells at different time points (as indicated in hours) by immunoblotting with antibodies against ORC1, RB, SUV39H1, CDC6, Cyclin E, ORC3 and a-Tubulin. ORC, Origin Recognition Complex; RB, Retinoblastoma. DOI: 10.7554/eLife.12785.012

    Article Snippet: For immunoblots, monoclonal FLAG antibody (F1804; Sigma), polyclonal GFP antibody (G1544; Sigma), monoclonal mouse SUV39H1 antibody (05–615; Millipore), monoclonal mouse T7 antibody (Cold Spring Harbor Laboratory antibody facility), mouse monoclonal anti-ORC1 antibody (pKS1-40) (Hemerly et al., 2009), monoclonal mouse ORC2 antibody (920-2-44) (Siddiqui and Stillman, 2007), mouse monoclonal ORC3 antibody (PKS1-16) (Prasanth et al., 2004), goat polyclonal anti-ORC4 antibody (ab9641; Abcam, Cambridge, MA), monoclonal mouse E2F-1 antibody (KH95; Santa Cruz Biotechnology, Dallas, TX) and rabbit GAL4 antibody (sc-577; Santa Cruz Biotechnology, Dallas, TX) were used.

    Techniques: Western Blot

    Figure 5. CDC6 co-operates with Cyclin E-CDK2 to activate E2F1-dependent CCNE1 gene transcription. (A–C) Equimolar amounts of MBP-GFP-ORC1 and MBP-RB proteins were incubated with increasing amounts of GST-CDC6 and/or Cyclin E-CDK2. MBP-GFP-ORC1 protein was immunoprecipitated with GFP antibody, then immunoblotted with the indicated antibodies. The purified proteins used in these experiments are shown in Figure 5—figure supplement 1 (A) MBP-GFP-ORC1 binds GST-CDC6. MBP protein served as control. (B) The binding of MBP-GFP-ORC1 protein to either GST-CCD6 in Figure 5 continued on next page

    Journal: eLife

    Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

    doi: 10.7554/elife.12785

    Figure Lengend Snippet: Figure 5. CDC6 co-operates with Cyclin E-CDK2 to activate E2F1-dependent CCNE1 gene transcription. (A–C) Equimolar amounts of MBP-GFP-ORC1 and MBP-RB proteins were incubated with increasing amounts of GST-CDC6 and/or Cyclin E-CDK2. MBP-GFP-ORC1 protein was immunoprecipitated with GFP antibody, then immunoblotted with the indicated antibodies. The purified proteins used in these experiments are shown in Figure 5—figure supplement 1 (A) MBP-GFP-ORC1 binds GST-CDC6. MBP protein served as control. (B) The binding of MBP-GFP-ORC1 protein to either GST-CCD6 in Figure 5 continued on next page

    Article Snippet: For immunoblots, monoclonal FLAG antibody (F1804; Sigma), polyclonal GFP antibody (G1544; Sigma), monoclonal mouse SUV39H1 antibody (05–615; Millipore), monoclonal mouse T7 antibody (Cold Spring Harbor Laboratory antibody facility), mouse monoclonal anti-ORC1 antibody (pKS1-40) (Hemerly et al., 2009), monoclonal mouse ORC2 antibody (920-2-44) (Siddiqui and Stillman, 2007), mouse monoclonal ORC3 antibody (PKS1-16) (Prasanth et al., 2004), goat polyclonal anti-ORC4 antibody (ab9641; Abcam, Cambridge, MA), monoclonal mouse E2F-1 antibody (KH95; Santa Cruz Biotechnology, Dallas, TX) and rabbit GAL4 antibody (sc-577; Santa Cruz Biotechnology, Dallas, TX) were used.

    Techniques: Incubation, Immunoprecipitation, Purification, Control, Binding Assay