Structured Review

Developmental Studies Hybridoma Bank mab anti islet 1
OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the <t>anti-islet-1</t> mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.
Mab Anti Islet 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mab anti islet 1/product/Developmental Studies Hybridoma Bank
Average 80 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mab anti islet 1 - by Bioz Stars, 2020-07
80/100 stars

Images

1) Product Images from "Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation"

Article Title: Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.17-11-04243.1997

OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.
Figure Legend Snippet: OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

Techniques Used: Staining, Expressing

2) Product Images from "Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation"

Article Title: Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.17-11-04243.1997

OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.
Figure Legend Snippet: OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

Techniques Used: Staining, Expressing

Related Articles

IA:

Article Title: Trio Controls the Mature Organization of Neuronal Clusters in the Hindbrain
Article Snippet: .. Sections were incubated with the monoclonal anti-Islet-1 (1:100, 39.4D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) or the polyclonal anti-Peripherin (1:200; Chemicon, Temecula, CA) antibodies overnight at room temperature. .. These primary antibodies were revealed using secondary anti-rabbit IgG antibodies conjugated to Alexa 488 (1:400; Invitrogen, Carlsbad, CA) or cyanine 3 (1:200; Jackson ImmunoResearch, West Grove, PA).

Article Title: Motoneuron-Derived Neurotrophin-3 Is a Survival Factor for PAX2-Expressing Spinal Interneurons
Article Snippet: .. Primary antibodies used were as follows: rabbit anti-PAX2 (1:200; Zymed, San Francisco, CA), polyclonal goat anti-choline acetyltransferase (AB144, 1:1000;Chemicon), monoclonal anti-Islet-1 (1:100, clone 4D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). .. Secondary antibodies were carboxymethyl indocyanine-3 (CY3)-goat anti-mouse IgG (1:200), Texas Red-donkey anti-goat IgG (1:200), and FITC-goat anti-rabbit IgG (1:200) (Jackson ImmunoResearch, West Grove, PA).

Incubation:

Article Title: Trio Controls the Mature Organization of Neuronal Clusters in the Hindbrain
Article Snippet: .. Sections were incubated with the monoclonal anti-Islet-1 (1:100, 39.4D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) or the polyclonal anti-Peripherin (1:200; Chemicon, Temecula, CA) antibodies overnight at room temperature. .. These primary antibodies were revealed using secondary anti-rabbit IgG antibodies conjugated to Alexa 488 (1:400; Invitrogen, Carlsbad, CA) or cyanine 3 (1:200; Jackson ImmunoResearch, West Grove, PA).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 80
    Developmental Studies Hybridoma Bank mab anti islet 1
    OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the <t>anti-islet-1</t> mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.
    Mab Anti Islet 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mab anti islet 1/product/Developmental Studies Hybridoma Bank
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mab anti islet 1 - by Bioz Stars, 2020-07
    80/100 stars
      Buy from Supplier

    80
    Developmental Studies Hybridoma Bank monoclonal anti islet 1 antibody
    Bar graphs showing the results of transplantation of cervical segments to the cervical region (control) and to the brachial region. A , The number (mean ± SD) of pyknotic cells at E4.5 after experimental (cervical to brachial transplant) and control (cervical to cervical transplant) transplants was not significantly different. B , The number of <t>Islet-1-immunopositive</t> neurons in the ventral horn in 10-μm-thick section at E5 was not significantly different between control and experimental groups. C , Transplantation of the cervical neural tube to the brachial region resulted in approximately 30% more Islet-1-immunopositive neurons on E9. p
    Monoclonal Anti Islet 1 Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti islet 1 antibody/product/Developmental Studies Hybridoma Bank
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti islet 1 antibody - by Bioz Stars, 2020-07
    80/100 stars
      Buy from Supplier

    86
    Developmental Studies Hybridoma Bank monoclonal mouse anti isl1
    <t>Isl1</t> and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes. A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p
    Monoclonal Mouse Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal mouse anti isl1/product/Developmental Studies Hybridoma Bank
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal mouse anti isl1 - by Bioz Stars, 2020-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

    doi: 10.1523/JNEUROSCI.17-11-04243.1997

    Figure Lengend Snippet: OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Article Snippet: The mAb anti-Islet-1, developed by Prof. T. M. Jessell, was obtained by the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine (Baltimore, MD), and the Department of Biological Sciences, University of Iowa (Iowa City, IA), under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development.

    Techniques: Staining, Expressing

    Bar graphs showing the results of transplantation of cervical segments to the cervical region (control) and to the brachial region. A , The number (mean ± SD) of pyknotic cells at E4.5 after experimental (cervical to brachial transplant) and control (cervical to cervical transplant) transplants was not significantly different. B , The number of Islet-1-immunopositive neurons in the ventral horn in 10-μm-thick section at E5 was not significantly different between control and experimental groups. C , Transplantation of the cervical neural tube to the brachial region resulted in approximately 30% more Islet-1-immunopositive neurons on E9. p

    Journal: The Journal of Neuroscience

    Article Title: A Novel Type of Programmed Neuronal Death in the Cervical Spinal Cord of the Chick Embryo

    doi: 10.1523/JNEUROSCI.16-11-03685.1996

    Figure Lengend Snippet: Bar graphs showing the results of transplantation of cervical segments to the cervical region (control) and to the brachial region. A , The number (mean ± SD) of pyknotic cells at E4.5 after experimental (cervical to brachial transplant) and control (cervical to cervical transplant) transplants was not significantly different. B , The number of Islet-1-immunopositive neurons in the ventral horn in 10-μm-thick section at E5 was not significantly different between control and experimental groups. C , Transplantation of the cervical neural tube to the brachial region resulted in approximately 30% more Islet-1-immunopositive neurons on E9. p

    Article Snippet: Monoclonal anti-Islet-1 antibody (40.2D6) was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), and the Department of Biology, University of Iowa (Iowa City, IA).

    Techniques: Transplantation Assay

    Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes. A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes. A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Expressing, Luciferase, Transfection

    EMSA with Isl1 truncations and Pou4f2. Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: EMSA with Isl1 truncations and Pou4f2. Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques:

    Pou4f2 and Isl1 bind to DNA elements individually and as a complex in EMSA. A: Wild-type (SBRN3(W)) sequence from the conserved element in the Sonic Hedgehog gene and its mutant sequence SBRN3(M) are shown on the top. Lines on top and beneath the SBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively. At Bottom, EMSA shows that either Pou4f2 or Isl1 alone can bind to SBRN3(W) as indicated by the slow migrating DNA-protein complexes, but not to SBRN3(M). When both Pou4f2 and Isl1 are present, they form a further slow-migrating DNA-protein complex with SBRN3(W), but not with SBRN3(M). B: Similarly, Pou4f2 or Isl1 alone binds to CBRN3(W), but not to CBRN3(M); Pou4f2 and Isl1 form a complex on CBRN3(W), but not on CBRN3(M). F indicates free probes. Lines on top and beneath the CBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Pou4f2 and Isl1 bind to DNA elements individually and as a complex in EMSA. A: Wild-type (SBRN3(W)) sequence from the conserved element in the Sonic Hedgehog gene and its mutant sequence SBRN3(M) are shown on the top. Lines on top and beneath the SBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively. At Bottom, EMSA shows that either Pou4f2 or Isl1 alone can bind to SBRN3(W) as indicated by the slow migrating DNA-protein complexes, but not to SBRN3(M). When both Pou4f2 and Isl1 are present, they form a further slow-migrating DNA-protein complex with SBRN3(W), but not with SBRN3(M). B: Similarly, Pou4f2 or Isl1 alone binds to CBRN3(W), but not to CBRN3(M); Pou4f2 and Isl1 form a complex on CBRN3(W), but not on CBRN3(M). F indicates free probes. Lines on top and beneath the CBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Sequencing, Mutagenesis, Binding Assay

    Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction. A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction. A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Positive Control

    Conserved DNA motifs in Ebf3 and Irx6 are recognized by Isl1/Pou4f2 complex. A: Mouse-human comparison to identify conserved regions in Ebf3 and Irx6 by VISTA. Red marks conserved non-coding region, light-blue is UTR, and dark-blue indicates exons. Heights of peaks indicate percentage of identify. The regions where the potential Isl1/Pou4f2 binding sites were identified are underlined. B: EMSA with wild-type and mutant probes derived from Ebf3 . The sequences of wild-type and mutant probes are shown on the top. Lines on top and underneath the wild-type sequences indicate the ATTA cores and Pou4f2 binding motif respectively. The bottom is the EMSA results. Both Isl1 and Pou4f2 could independently bind to the wild-type probe, but not the mutant probe. The Isl1/Pou4f2 complex can bind the wild-type probe, but not the mutant, as indicated by the slow-migrating complex. C: The same experiment as that of B, except that the wild-type and mutant probes were derived from Irx6 . Both proteins as well as the complex could bind to the wild-type probe, but not the mutant one. D: ChIP analysis of Isl1 and Pou4f2 binding in vivo. Genomic DNA from ChIP by anti-Pou4f2 (P4f2) and anti-Isl1 were amplified by PCR with primers flanking the Isl1/Pou4f2-binding regions of Ebf3 and Isl1 and a control region of β-actin. Non-specific IgG was used as control in both ChIP experiments.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Conserved DNA motifs in Ebf3 and Irx6 are recognized by Isl1/Pou4f2 complex. A: Mouse-human comparison to identify conserved regions in Ebf3 and Irx6 by VISTA. Red marks conserved non-coding region, light-blue is UTR, and dark-blue indicates exons. Heights of peaks indicate percentage of identify. The regions where the potential Isl1/Pou4f2 binding sites were identified are underlined. B: EMSA with wild-type and mutant probes derived from Ebf3 . The sequences of wild-type and mutant probes are shown on the top. Lines on top and underneath the wild-type sequences indicate the ATTA cores and Pou4f2 binding motif respectively. The bottom is the EMSA results. Both Isl1 and Pou4f2 could independently bind to the wild-type probe, but not the mutant probe. The Isl1/Pou4f2 complex can bind the wild-type probe, but not the mutant, as indicated by the slow-migrating complex. C: The same experiment as that of B, except that the wild-type and mutant probes were derived from Irx6 . Both proteins as well as the complex could bind to the wild-type probe, but not the mutant one. D: ChIP analysis of Isl1 and Pou4f2 binding in vivo. Genomic DNA from ChIP by anti-Pou4f2 (P4f2) and anti-Isl1 were amplified by PCR with primers flanking the Isl1/Pou4f2-binding regions of Ebf3 and Isl1 and a control region of β-actin. Non-specific IgG was used as control in both ChIP experiments.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Binding Assay, Mutagenesis, Derivative Assay, Chromatin Immunoprecipitation, In Vivo, Amplification, Polymerase Chain Reaction

    GST pull-down assays show other Lim-Homeodomain and POU-domain proteins interact with Pou4f2 and Isl1 respectively. A: GST-Isl1 (GST-I), but not GST alone, can interact with Pou3f2 and Pou4f3, but not Pou6f1. Pou4f2 was used as positive control. Top: Detection of pulled down proteins by anti-His. Middle: Input GST or GST-Isl1 by anti-GST. Bottom: Input POU-Homeodmain proteins detected by anti-His. B,C,D: GST-Pou4f2 (GST-P), but not GST alone, can interact with Isl2, Lhx2, but not Lhx1. In each panel, the top is detection of pulled down protein by the indicated antibodies, the middle is input GST or GST-Pou4f2 as detected by anti-GST, and the bottom is detection of the input Lim-Homeodomain proteins by the indicated antibodies (anti-Isl1 for Isl1 and Isl2, anti-Lhx2 for Lhx2, and anti-His for Lhx1).

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: GST pull-down assays show other Lim-Homeodomain and POU-domain proteins interact with Pou4f2 and Isl1 respectively. A: GST-Isl1 (GST-I), but not GST alone, can interact with Pou3f2 and Pou4f3, but not Pou6f1. Pou4f2 was used as positive control. Top: Detection of pulled down proteins by anti-His. Middle: Input GST or GST-Isl1 by anti-GST. Bottom: Input POU-Homeodmain proteins detected by anti-His. B,C,D: GST-Pou4f2 (GST-P), but not GST alone, can interact with Isl2, Lhx2, but not Lhx1. In each panel, the top is detection of pulled down protein by the indicated antibodies, the middle is input GST or GST-Pou4f2 as detected by anti-GST, and the bottom is detection of the input Lim-Homeodomain proteins by the indicated antibodies (anti-Isl1 for Isl1 and Isl2, anti-Lhx2 for Lhx2, and anti-His for Lhx1).

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Positive Control

    Pou4f2 and Isl1 form a complex in vitro and in vivo. A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Pou4f2 and Isl1 form a complex in vitro and in vivo. A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: In Vitro, In Vivo, Immunoprecipitation

    Diagram of serial deletions of Pou4f2 and Isl1. Diagram of the Pou4f2 (Left) and Isl1 (right) protein structures and the truncated deletions we expressed in E. Coli . The amino acid residues for each deletion and their estimated molecular weights are indicated. All proteins are His-tagged at the C terminus for easy detection by Western Blotting, although the Isl1 protein and its truncates are detected by a polyclonal rabbit anti-Isl1. * indicates constructs capable of protein-protein interaction (see Figs. 3 5 ).

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Diagram of serial deletions of Pou4f2 and Isl1. Diagram of the Pou4f2 (Left) and Isl1 (right) protein structures and the truncated deletions we expressed in E. Coli . The amino acid residues for each deletion and their estimated molecular weights are indicated. All proteins are His-tagged at the C terminus for easy detection by Western Blotting, although the Isl1 protein and its truncates are detected by a polyclonal rabbit anti-Isl1. * indicates constructs capable of protein-protein interaction (see Figs. 3 5 ).

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Western Blot, Construct

    EMSA with Pou4f2 truncations and Isl1. Left: Isl1 forms complexes with Pou4f2D4 and Pou4f2D5 on SBRN3(W), but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3. Right: Isl1 forms complexes with Pou4f2 D4 and Pou4f2D5, but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3 on CBRN(W). F is free probe. These results are consistent with those from the pull-down assays.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: EMSA with Pou4f2 truncations and Isl1. Left: Isl1 forms complexes with Pou4f2D4 and Pou4f2D5 on SBRN3(W), but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3. Right: Isl1 forms complexes with Pou4f2 D4 and Pou4f2D5, but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3 on CBRN(W). F is free probe. These results are consistent with those from the pull-down assays.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: