Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Defective eye development in adult Egr1−/− BALB/c mice. (A and B) External view of normal eyes from adult Egr1+/−, Egr1−/− C57BL/6 mice (A) and adult Egr1+/+, Egr1+/−BALB/c mice (B). (C) External view of eyes from adult Egr1−/− BALB/c mice showing abnormal periocular shape and eye size and corneal opacity. (D) Histology of adult Egr1+/+ BALB/c mice. (E–G) Histology of adult Egr1−/− BALB/c adult mice at F5 from the initial backcross, showing anophthalmia (E), severe microphthalmia (F), and microphthalmia (G). (H–J) Abnormalities in adult Egr1−/− BALB/c mice. In G–J, numbered arrows indicate the following: 1, retinal dysplasia; 2, cataract; 3, calcification; 4, corneal neovascularization; 5, keratitis. (Scale bars: D, 800 µm; E and F, 400 µm; G and H, 200 µm; I, 100 µm; J, 50 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of adult Egr1−/− BALB/c mice at F4 from the second backcross. (A) Anophthalmia. (B) Severe microphthalmia. (C) Retinal rosettes and folds. (D) Keratitis and synechiae (adhesion of lens and cornea). Numbered arrows indicate (1) retinal dysplasia; (2) retinal folds; (3) cataract; (4) lens capsule rupture. (Scale bars: A and B, 400 µm; C and D, 200 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Abnormal ocular development in postnatal Egr1−/− BALB/c mice. (A–G) Histology of eyes from newborn Egr1+/− mice (A), Egr1−/− mice at postnatal day 1 (B) and 2 (C), and from Egr1−/− mice at postnatal days 3 and 4 (D–G). In B and D–G, numbered arrows refer to the following: 1, mild corneal neovascularization; 2, mild keratitis; 3, cataracts; 4, corneal perforation; 5, vitreous hemorrhage; 6, retinal detachment; 7, inflammation; 8, residual remnant retina. (H–J) Eyelids of newborn Egr1+/+ (H) and Egr1+/− (I) mice are formed, whereas those of Egr1−/− mice are not formed, and eyes appear open (J). [Scale bars: A, B (Left), and D, 800 µm; C, 400 µm; E (Left), F, and G, 200 µm; B (Right) and E (Right), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Eyelid formation in embryos. (A, C, E) Egr1+/− BALB/c mice. (B, D, F) Egr1−/− BALB/c mice. Two embryos of each indicated genotype are shown at days E16.5 (A and B), E17.5 (C and D), and E18.5 (E and F). The arrows in A and B indicate areas of eyelid merging or defects therein. (Scale bars: A–F, 800 μm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Immunohistochemistry of Egr1 during embryogenesis. Two embryos of C57BL/6 WT mice at days E13.5 (A and B), E15.5 (C and D), and E17.5 (E and F) and of BALB/c WT mice at day E15.5 (G and H). A, C, E, and G show controls stained with the Egr1 Ab-blocking peptide mix, and B, D, F, and H show staining with the Egr1 antibody. In B, D, F, and H, Left are the same magnification as in panels A, C, E, and F, respectively, whereas B, D, F, and H, Right show a higher-magnification view of the relevant region of the left panel. [Scale bars: A, B (Left), C, D (Left), E, F (Left), G, and H (Left), 200 µm; B (Right), D (Right), F (Right), and H (Right), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques: Immunohistochemistry, Staining, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Gene-expression profiles in eyes from Egr1+/− and Egr1−/− BALB/c mice. (A) Il1b mRNA expression is significantly increased in adult Egr1−/− BALB/c but not in Egr1−/−C57BL/6 mice. (B) Il1b mRNA is increased in Egr1−/− BALB/c newborn mice. RNA was normalized relative to the expression of Rpl7. Comparison between samples was done by the Student’s t test. (C–G) Gene-expression profiles in eyes from Egr1+/− and Egr1−/− BALB/c mice at postnatal day 3. (C) MDS analysis for individual samples from eyes from Egr1+/− and Egr1−/− mice with formed eyelids (indicated by a “C” for closed) vs. Egr1−/− mice without eyelid formation (indicated by an “O” for open). Differences in dimension 1 (Dim1, x axis) indicates greater differences in samples than do differences in dimension 2 (Dim 2, y axis). (D) 2D scatter plot shows expressed genes. Genes highlighted in blue, green, and orange indicate differentially expressed genes with |log2FC|>3, (|log2FC|>2), and |log2FC|>1, respectively. “log2FC” refers to the log (base 2) of the fold change of mRNA expression in eyes with unformed eyelids vs. eyes with formed eyelids. log2CPM, log base 2 of counts per million. (E) Heat map of the top 50 differentially expressed genes. The expression matrix on rows is normalized by z-scores, and the colors of the heat map are mapped linearly to the z-scores. Samples ending in “O” were from open eyes of a given embryo; those ending in “C” were from closed eyes. (F) Volcano plot showing the top 50 differentially expressed genes. The x axis represents the log2FC between eyes from Egr1−/− mice with unformed eyelids (open) vs. Egr1−/− or Egr1+/− mice with fused eyelids (closed), and the y axis shows the −log10 of the P value. (G) Bar plots show the expression of Il1b, Krt16, Sprr1a, and Egr1. OD, right eye; OS, left eye. E24, E28, E29, and E32 are the originally designations for these embryos based on when they were collected. RPKM, reads per kilobase of transcript per million mapped reads.

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Eye phenotype of Il1r tm1Imx Egr1 −/− BALB/c double KO mice

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of Egr1−/−, Tyrc-2J/c-2J C57BL/6 mice. (A) Normal structure of eyes from Egr1−/−, Tyr+/c-2J C57BL/6 adult mice. (B–D) Abnormalities in Egr1−/−, Tyr c-2J/c-2J C57BL/6 adult mice. (B) Mild corneal neovascularization (1). (C) Poor corneal closure with epithelial ingrowth (2), retinal dysplasia (3), anterior angle dysplasia and ciliary body hypoplasia (4), and PHPV (5). The left and upper panels are higher- magnification views of the indicated areas. (D) Staphyloma, posterior coloboma, and PHPV. [Scale bars: A, C (Bottom), and D, 800 µm; B, 50 µm; C (Left and Top), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histological analysis of Egr1 −/− Tyr c-2J C57BL/6 mice

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of eyes in Egr1+/− BALB/c (A) and Egr1−/− BALB/c (B) embryos at day E14.5. (Scale bars: A and B, 100 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Defective eye development in adult Egr1−/− BALB/c mice. (A and B) External view of normal eyes from adult Egr1+/−, Egr1−/− C57BL/6 mice (A) and adult Egr1+/+, Egr1+/−BALB/c mice (B). (C) External view of eyes from adult Egr1−/− BALB/c mice showing abnormal periocular shape and eye size and corneal opacity. (D) Histology of adult Egr1+/+ BALB/c mice. (E–G) Histology of adult Egr1−/− BALB/c adult mice at F5 from the initial backcross, showing anophthalmia (E), severe microphthalmia (F), and microphthalmia (G). (H–J) Abnormalities in adult Egr1−/− BALB/c mice. In G–J, numbered arrows indicate the following: 1, retinal dysplasia; 2, cataract; 3, calcification; 4, corneal neovascularization; 5, keratitis. (Scale bars: D, 800 µm; E and F, 400 µm; G and H, 200 µm; I, 100 µm; J, 50 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of adult Egr1−/− BALB/c mice at F4 from the second backcross. (A) Anophthalmia. (B) Severe microphthalmia. (C) Retinal rosettes and folds. (D) Keratitis and synechiae (adhesion of lens and cornea). Numbered arrows indicate (1) retinal dysplasia; (2) retinal folds; (3) cataract; (4) lens capsule rupture. (Scale bars: A and B, 400 µm; C and D, 200 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Abnormal ocular development in postnatal Egr1−/− BALB/c mice. (A–G) Histology of eyes from newborn Egr1+/− mice (A), Egr1−/− mice at postnatal day 1 (B) and 2 (C), and from Egr1−/− mice at postnatal days 3 and 4 (D–G). In B and D–G, numbered arrows refer to the following: 1, mild corneal neovascularization; 2, mild keratitis; 3, cataracts; 4, corneal perforation; 5, vitreous hemorrhage; 6, retinal detachment; 7, inflammation; 8, residual remnant retina. (H–J) Eyelids of newborn Egr1+/+ (H) and Egr1+/− (I) mice are formed, whereas those of Egr1−/− mice are not formed, and eyes appear open (J). [Scale bars: A, B (Left), and D, 800 µm; C, 400 µm; E (Left), F, and G, 200 µm; B (Right) and E (Right), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Eyelid formation in embryos. (A, C, E) Egr1+/− BALB/c mice. (B, D, F) Egr1−/− BALB/c mice. Two embryos of each indicated genotype are shown at days E16.5 (A and B), E17.5 (C and D), and E18.5 (E and F). The arrows in A and B indicate areas of eyelid merging or defects therein. (Scale bars: A–F, 800 μm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Immunohistochemistry of Egr1 during embryogenesis. Two embryos of C57BL/6 WT mice at days E13.5 (A and B), E15.5 (C and D), and E17.5 (E and F) and of BALB/c WT mice at day E15.5 (G and H). A, C, E, and G show controls stained with the Egr1 Ab-blocking peptide mix, and B, D, F, and H show staining with the Egr1 antibody. In B, D, F, and H, Left are the same magnification as in panels A, C, E, and F, respectively, whereas B, D, F, and H, Right show a higher-magnification view of the relevant region of the left panel. [Scale bars: A, B (Left), C, D (Left), E, F (Left), G, and H (Left), 200 µm; B (Right), D (Right), F (Right), and H (Right), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques: Immunohistochemistry, Staining, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Gene-expression profiles in eyes from Egr1+/− and Egr1−/− BALB/c mice. (A) Il1b mRNA expression is significantly increased in adult Egr1−/− BALB/c but not in Egr1−/−C57BL/6 mice. (B) Il1b mRNA is increased in Egr1−/− BALB/c newborn mice. RNA was normalized relative to the expression of Rpl7. Comparison between samples was done by the Student’s t test. (C–G) Gene-expression profiles in eyes from Egr1+/− and Egr1−/− BALB/c mice at postnatal day 3. (C) MDS analysis for individual samples from eyes from Egr1+/− and Egr1−/− mice with formed eyelids (indicated by a “C” for closed) vs. Egr1−/− mice without eyelid formation (indicated by an “O” for open). Differences in dimension 1 (Dim1, x axis) indicates greater differences in samples than do differences in dimension 2 (Dim 2, y axis). (D) 2D scatter plot shows expressed genes. Genes highlighted in blue, green, and orange indicate differentially expressed genes with |log2FC|>3, (|log2FC|>2), and |log2FC|>1, respectively. “log2FC” refers to the log (base 2) of the fold change of mRNA expression in eyes with unformed eyelids vs. eyes with formed eyelids. log2CPM, log base 2 of counts per million. (E) Heat map of the top 50 differentially expressed genes. The expression matrix on rows is normalized by z-scores, and the colors of the heat map are mapped linearly to the z-scores. Samples ending in “O” were from open eyes of a given embryo; those ending in “C” were from closed eyes. (F) Volcano plot showing the top 50 differentially expressed genes. The x axis represents the log2FC between eyes from Egr1−/− mice with unformed eyelids (open) vs. Egr1−/− or Egr1+/− mice with fused eyelids (closed), and the y axis shows the −log10 of the P value. (G) Bar plots show the expression of Il1b, Krt16, Sprr1a, and Egr1. OD, right eye; OS, left eye. E24, E28, E29, and E32 are the originally designations for these embryos based on when they were collected. RPKM, reads per kilobase of transcript per million mapped reads.

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Eye phenotype of Il1r tm1Imx Egr1 −/− BALB/c double KO mice

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of Egr1−/−, Tyrc-2J/c-2J C57BL/6 mice. (A) Normal structure of eyes from Egr1−/−, Tyr+/c-2J C57BL/6 adult mice. (B–D) Abnormalities in Egr1−/−, Tyr c-2J/c-2J C57BL/6 adult mice. (B) Mild corneal neovascularization (1). (C) Poor corneal closure with epithelial ingrowth (2), retinal dysplasia (3), anterior angle dysplasia and ciliary body hypoplasia (4), and PHPV (5). The left and upper panels are higher- magnification views of the indicated areas. (D) Staphyloma, posterior coloboma, and PHPV. [Scale bars: A, C (Bottom), and D, 800 µm; B, 50 µm; C (Left and Top), 100 µm.]

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histological analysis of Egr1 −/− Tyr c-2J C57BL/6 mice

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genetic background-dependent role of Egr1 for eyelid development

doi: 10.1073/pnas.1705848114

Figure Lengend Snippet: Histology of eyes in Egr1+/− BALB/c (A) and Egr1−/− BALB/c (B) embryos at day E14.5. (Scale bars: A and B, 100 µm.)

Article Snippet: Following a 5% normal goat serum block, three sets of slides were incubated overnight at 4 °C with ( i ) Egr1 Ab (rabbit mAb) (no. 4153; Cell Signaling Technology) at 1:100, ( ii ) Egr1 Ab/blocking peptide (no. 1015; Cell Signaling Technology) mix (1:2 by volume), and ( iii ) rabbit monoclonal IgG isotype control reagent (no. 3900; Cell Signaling Technology).

Techniques:

Journal: British microbiology research journal

Article Title: Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

doi: 10.9734/BMRJ/2013/4817

Figure Lengend Snippet: Construction of recombinant virus over-expressing Egr-1 and its regulatory effects on HSV-1.(a). Schematic representation of recombinant virus construction. (b). Infection of SIRC cells followed by RNA isolation and qRT-PCR using primers against open reading frames of Egr-1 and ICP0.(c). The same experiments described in 4b were performed to analyze ICP0 expression profile

Article Snippet: Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with pre-immune IgG (Cat#: 2729; Cell Signaling; Boston, MA) as control followed by addition of anti-Egr-1 Ab (Cat#: 4153; Cell Signaling; Boston, MA).

Techniques: Recombinant, Expressing, Infection, Isolation, Quantitative RT-PCR

Journal: British microbiology research journal

Article Title: Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

doi: 10.9734/BMRJ/2013/4817

Figure Lengend Snippet: Egr-1 induced by HSV-1 infection required active viral gene expression (a). SIRC cells were infected by 17 syn+ EGFP strains of HSV-1 at 4°C or 37°C and subjected to Western blot analyses at 24 hpi using antibodies against Egr-1, GFP (infection control), and α-Tubulin (loading control).(b). Infection described in a. was performed using regular or UV-inactivated virus at 37°C and the cell lysates were collected at 24 hpi followed by Western blot analyses.(c). Same infection shown in a. and b. were performed analyzed by quantitative Cignal ™ Reporter Assay.(d). qRTPCR was performed to compare the accumulation of Egr-1 transcript at different time points (0.5, 1, 2, and 4 hpi). The amount of Egr-1 mRNA was normalized by two cellular genes PPIA and PGK1 which were reported not to be affected by HSV-1 infection (materials and methods)

Article Snippet: Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with pre-immune IgG (Cat#: 2729; Cell Signaling; Boston, MA) as control followed by addition of anti-Egr-1 Ab (Cat#: 4153; Cell Signaling; Boston, MA).

Techniques: Infection, Expressing, Western Blot, Reporter Assay

Journal: British microbiology research journal

Article Title: Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

doi: 10.9734/BMRJ/2013/4817

Figure Lengend Snippet: Egr-1 induction does not require viral replication. (a). Infections of SIRC with 17 syn+ EGFP strains of HSV-1 were performed in the presence or absence of ACV followed by Western blot analyses at 24 hpi using antibodies against Egr-1, GFP (infection control), and α-Tubulin. (b). Immunofluorescent microscopy was done at 24 hpi to validate the observation in a. Egr-1 was effectively produced even without the replication (compare c, g, k, and o). Green fluorescent signals represent the cells infected by viruses

Article Snippet: Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with pre-immune IgG (Cat#: 2729; Cell Signaling; Boston, MA) as control followed by addition of anti-Egr-1 Ab (Cat#: 4153; Cell Signaling; Boston, MA).

Techniques: Western Blot, Infection, Microscopy, Produced

Journal: British microbiology research journal

Article Title: Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

doi: 10.9734/BMRJ/2013/4817

Figure Lengend Snippet: HSV-1 IE regulatory sequences were bound by induced Egr-1. (a). Infections of SIRC cells at moi of 5 with a receptor tyrosine kinase inhibitor AG1007 at 1μM were compared to infection without the inhibitor by Western Blot analyses.(b). Same infections described in 3a were performed with or without AG1007 followed by ChIP via regular and qPCR using ICP22 primers against the EBE to address the Egr-1 occupancy. The binding of Egr-1 to ICP22 EBE was confirmed and the interaction was decreased to 42% by AG1007 treatment. (c). The Egr-1 occupancy on ICP0 promoter was analyzed by ChIP described in 3b. The quantitative PCR indicated that Egr-1 recruitment to ICP0 promoter was reduced to 28% by AG1007

Article Snippet: Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with pre-immune IgG (Cat#: 2729; Cell Signaling; Boston, MA) as control followed by addition of anti-Egr-1 Ab (Cat#: 4153; Cell Signaling; Boston, MA).

Techniques: Infection, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction

Journal: British microbiology research journal

Article Title: Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

doi: 10.9734/BMRJ/2013/4817

Figure Lengend Snippet: Egr-1 promoted HSV-1 replication and release of infectious viruses. The plaque assays were performed by infecting Vero cells using supernatant from infections of 293HEK and SIRC cells with wild-type and recombinant viruses

Article Snippet: Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with pre-immune IgG (Cat#: 2729; Cell Signaling; Boston, MA) as control followed by addition of anti-Egr-1 Ab (Cat#: 4153; Cell Signaling; Boston, MA).

Techniques: Recombinant

Journal: PLoS Biology

Article Title: A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

doi: 10.1371/journal.pbio.1000476

Figure Lengend Snippet: (a) TPO-dependent EGR1 induction is inhibited by PD98056 or U0126 treatment. UT711oc1 cells were cultured for 5 d in presence of TPO and two different MAPK inhibitors. (b) EGR1 increases after MEK1-SS/DD expression in presence of GM-CSF. (c) TPO induces the translocation of EGR1 to the nucleus. Cells were grown with GM-CSF or TPO for 2 h and lysates were fractionated before being resolved by Western blotting. (d) Immunolabeling of EGR1 (in red) and nucleus (in blue) were performed and analyzed by confocal microscopy. EGR1 was present in low quantity in cytoplasm in proliferating UT711oc1 (GM-CSF), but TPO induced an increase in EGR1 labeling and translocation of the transcription factor to the nucleus. (e) EGR1 shRNA lentiviral transductions efficiently repress EGR1 mRNA expression after TPO exposure and (f) down-regulate expression of p21. (g) EGR1 knock-down inhibits the SA-β-galactosidase staining. (h) Study of chromatin immunoprecipitation (ChIP) with EGR1 antibody shows enrichment in p21 promoter after TPO exposure compared to GM-CSF. The figures represent one of three performed experiments. Error bar represents the standard deviation of three independent experiments.

Article Snippet: Chromatin immunoprecipitations (ChIP) assays were performed using a ChIP assay kit (cell signaling) with anti-EGR1 Ab (cell signaling).

Techniques: Cell Culture, Expressing, Translocation Assay, Western Blot, Immunolabeling, Confocal Microscopy, Labeling, shRNA, Staining, Chromatin Immunoprecipitation, Standard Deviation