Structured Review

Epitomics anti islet 1
DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by <t>Islet-1.</t> (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P
Anti Islet 1, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1"

Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.6343

DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P
Figure Legend Snippet: DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P

Techniques Used: DNA Methylation Assay, Methylation, MSP Assay, Chromatin Immunoprecipitation

Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P
Figure Legend Snippet: Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P

Techniques Used: Transfection, Microscopy, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Infection

Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.
Figure Legend Snippet: Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

Techniques Used: Over Expression, Fluorescence, Microscopy, Infection, Flow Cytometry, Cytometry, Expressing, Western Blot, Binding Assay

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Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor
Article Snippet: .. The membrane was blocked in PBS/Tween supplemented with 5% nonfat dry milk followed by incubation overnight with anti-Ldb1 (1:2,000; see Supplementary Table 2 for additional antibody sources), anti-Isl1 (1:2,000), anti-Pdx1 (1:20,000), anti-Pax6 (1:1,000), anti-NeuroD1 (1:3,000; 3181-1; Epitomics), anti-Hnf1α (1:2,000, sc-6548X; Santa Cruz Biotechnology), and anti-MafA (1:2,000, A300 BL-1225; Bethyl Laboratories). .. The washed membrane was incubated with horseradish peroxidase-conjugated secondary antibody followed by detection using Western-Lightning Plus-ECL (PerkinElmer, Waltham, MA).

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    Epitomics anti islet 1
    DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by <t>Islet-1.</t> (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P
    Anti Islet 1, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti islet 1/product/Epitomics
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti islet 1 - by Bioz Stars, 2020-09
    91/100 stars
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    DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: DNA Methylation Assay, Methylation, MSP Assay, Chromatin Immunoprecipitation

    Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: Transfection, Microscopy, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Infection

    Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: Over Expression, Fluorescence, Microscopy, Infection, Flow Cytometry, Cytometry, Expressing, Western Blot, Binding Assay

    E18.5 Glut2 and Hb9 mRNA and protein expression is only compromised in Ldb1 mutant mice. Immunofluorescence analysis of Glut2 (red) and insulin (green) in the E18.5 control, mutant Ldb1 ( A ), and mutant Isl1 ( B ) pancreas. Insets show magnified insulin + cell clusters. C : ChIP-Seq pictograph demonstrating Isl1 occupancy at distal Glut2 Re1 (5′) and Re2 (3′) domains in βTC-3 cells. The red line denotes the Glut2-coding region, whereas ChIP-tested proximal 5′ promoter region is represented by the red box. D : βTC-3 ChIP analysis of Isl1 ( top panel ) and Ldb1 ( bottom panel ) occupancy of Glut2 Re1, Re2, and the proximal domain compared with the PEPCK control (from top to bottom , respectively). H 2 O serves as a negative PCR control. Results recapitulate observed Isl1 ChIP-Seq occupation of Glut2 Re1 and Re2, whereas Ldb1 also binds to the proximal domain. E : qPCR analysis of E18.5 Hb9 mRNA levels in pancreata from Ldb1- (blue bar) and Isl1-deficient (red bar) pancreata. Littermate control mRNA level was set at onefold (dashed line) ± SEM. F : E18.5 immunostaining analysis demonstrates that Hb9 protein (white) is maintained in the insulin + (red) nuclei of Pdx1-Cre ; Isl1 F/F pancreata as compared with littermate controls, as denoted by the white arrowheads. G : However, Hb9 is lost from most remaining insulin + cells in the Pax6-Cre ; Ldb1 F/F pancreata seen by comparing white arrowhead–labeled Hb9 + cells of control and mutant in F and G . The nuclear Hb9 signals are shown in the right panels . Yellow arrowheads in G illustrate autofluorescence from erythrocytes. * P

    Journal: Diabetes

    Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor

    doi: 10.2337/db12-0952

    Figure Lengend Snippet: E18.5 Glut2 and Hb9 mRNA and protein expression is only compromised in Ldb1 mutant mice. Immunofluorescence analysis of Glut2 (red) and insulin (green) in the E18.5 control, mutant Ldb1 ( A ), and mutant Isl1 ( B ) pancreas. Insets show magnified insulin + cell clusters. C : ChIP-Seq pictograph demonstrating Isl1 occupancy at distal Glut2 Re1 (5′) and Re2 (3′) domains in βTC-3 cells. The red line denotes the Glut2-coding region, whereas ChIP-tested proximal 5′ promoter region is represented by the red box. D : βTC-3 ChIP analysis of Isl1 ( top panel ) and Ldb1 ( bottom panel ) occupancy of Glut2 Re1, Re2, and the proximal domain compared with the PEPCK control (from top to bottom , respectively). H 2 O serves as a negative PCR control. Results recapitulate observed Isl1 ChIP-Seq occupation of Glut2 Re1 and Re2, whereas Ldb1 also binds to the proximal domain. E : qPCR analysis of E18.5 Hb9 mRNA levels in pancreata from Ldb1- (blue bar) and Isl1-deficient (red bar) pancreata. Littermate control mRNA level was set at onefold (dashed line) ± SEM. F : E18.5 immunostaining analysis demonstrates that Hb9 protein (white) is maintained in the insulin + (red) nuclei of Pdx1-Cre ; Isl1 F/F pancreata as compared with littermate controls, as denoted by the white arrowheads. G : However, Hb9 is lost from most remaining insulin + cells in the Pax6-Cre ; Ldb1 F/F pancreata seen by comparing white arrowhead–labeled Hb9 + cells of control and mutant in F and G . The nuclear Hb9 signals are shown in the right panels . Yellow arrowheads in G illustrate autofluorescence from erythrocytes. * P

    Article Snippet: The membrane was blocked in PBS/Tween supplemented with 5% nonfat dry milk followed by incubation overnight with anti-Ldb1 (1:2,000; see Supplementary Table 2 for additional antibody sources), anti-Isl1 (1:2,000), anti-Pdx1 (1:20,000), anti-Pax6 (1:1,000), anti-NeuroD1 (1:3,000; 3181-1; Epitomics), anti-Hnf1α (1:2,000, sc-6548X; Santa Cruz Biotechnology), and anti-MafA (1:2,000, A300 BL-1225; Bethyl Laboratories).

    Techniques: Expressing, Mutagenesis, Mouse Assay, Immunofluorescence, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunostaining, Labeling

    Glp1r is a novel Ldb1- and Isl1-activated target gene. A : qPCR quantification of Isl1 ChIP-Seq candidates from Ldb1- (blue bars) and Isl1-deficient (red bars) E18.5 pancreata ( n = 4–6). Data are presented as fold of the littermate control, which was set at 1 (marked by the dashed line), ± SEM. B and C : Immunostaining of Glp1r (brown) and insulin (red) at P6 illustrates reduced Glp1r protein levels in insulin + cells lacking Ldb1 or Isl1. D : βTC-3 ChIP-Seq pictograph demonstrating the four distal 5′ peaks of Isl1 occupancy near Glp1r . The red line denotes the Glp1r locus. E : ChIP enrichment of peak 1 Glp1r 5′ DNA in Isl1 ( top panel ) and Ldb1 βTC-3 immunoprecipitates ( bottom panel ) as compared with IgG control-treated DNA. H 2 O serves as a negative control for the PCR. * P

    Journal: Diabetes

    Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor

    doi: 10.2337/db12-0952

    Figure Lengend Snippet: Glp1r is a novel Ldb1- and Isl1-activated target gene. A : qPCR quantification of Isl1 ChIP-Seq candidates from Ldb1- (blue bars) and Isl1-deficient (red bars) E18.5 pancreata ( n = 4–6). Data are presented as fold of the littermate control, which was set at 1 (marked by the dashed line), ± SEM. B and C : Immunostaining of Glp1r (brown) and insulin (red) at P6 illustrates reduced Glp1r protein levels in insulin + cells lacking Ldb1 or Isl1. D : βTC-3 ChIP-Seq pictograph demonstrating the four distal 5′ peaks of Isl1 occupancy near Glp1r . The red line denotes the Glp1r locus. E : ChIP enrichment of peak 1 Glp1r 5′ DNA in Isl1 ( top panel ) and Ldb1 βTC-3 immunoprecipitates ( bottom panel ) as compared with IgG control-treated DNA. H 2 O serves as a negative control for the PCR. * P

    Article Snippet: The membrane was blocked in PBS/Tween supplemented with 5% nonfat dry milk followed by incubation overnight with anti-Ldb1 (1:2,000; see Supplementary Table 2 for additional antibody sources), anti-Isl1 (1:2,000), anti-Pdx1 (1:20,000), anti-Pax6 (1:1,000), anti-NeuroD1 (1:3,000; 3181-1; Epitomics), anti-Hnf1α (1:2,000, sc-6548X; Santa Cruz Biotechnology), and anti-MafA (1:2,000, A300 BL-1225; Bethyl Laboratories).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunostaining, Negative Control, Polymerase Chain Reaction

    Ldb1 is enriched in islet and ductal cells. A : Ldb1 ( left ) and Ldb2 ( middle ) mRNA expression was visualized by RNA ISH under identical conditions in E15.5 tissue. A higher-magnification view of pancreatic Ldb1 and Ldb2 expression is shown on the right . B : qPCR was performed to measure Ldb1 , Ldb2 , and Isl1 mRNA levels in E15.5 total pancreas (black bars) and 3-month-old isolated islets (gray bars). Expression levels are displayed relative to TATA-binding protein (TBP), which is set as onefold. Error bars represent ± SEM ( n = 5). Ldb1 mRNA is significantly more abundant than Ldb2 in E15.5 and adult samples. C – K : Ldb1, Pdx1, Isl1, hormone (insulin, glucagon, and somatostatin), and ductal (DBA, CK-19) markers were visualized at E10.5, E18.5, P5, and P21 by coimmunofluorescence. Yellow dashed lines mark dorsal and ventral pancreas domains in C and D . Notably, only a few of the pancreatic Ldb1 + cells in D are copositive for Isl1 at this stage (some marked by white arrowheads). L : Immunohistochemical analysis illustrates enriched Ldb1 protein (brown) expression in adult human islet cells; the sample is eosin (pink) counterstained. * P

    Journal: Diabetes

    Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor

    doi: 10.2337/db12-0952

    Figure Lengend Snippet: Ldb1 is enriched in islet and ductal cells. A : Ldb1 ( left ) and Ldb2 ( middle ) mRNA expression was visualized by RNA ISH under identical conditions in E15.5 tissue. A higher-magnification view of pancreatic Ldb1 and Ldb2 expression is shown on the right . B : qPCR was performed to measure Ldb1 , Ldb2 , and Isl1 mRNA levels in E15.5 total pancreas (black bars) and 3-month-old isolated islets (gray bars). Expression levels are displayed relative to TATA-binding protein (TBP), which is set as onefold. Error bars represent ± SEM ( n = 5). Ldb1 mRNA is significantly more abundant than Ldb2 in E15.5 and adult samples. C – K : Ldb1, Pdx1, Isl1, hormone (insulin, glucagon, and somatostatin), and ductal (DBA, CK-19) markers were visualized at E10.5, E18.5, P5, and P21 by coimmunofluorescence. Yellow dashed lines mark dorsal and ventral pancreas domains in C and D . Notably, only a few of the pancreatic Ldb1 + cells in D are copositive for Isl1 at this stage (some marked by white arrowheads). L : Immunohistochemical analysis illustrates enriched Ldb1 protein (brown) expression in adult human islet cells; the sample is eosin (pink) counterstained. * P

    Article Snippet: The membrane was blocked in PBS/Tween supplemented with 5% nonfat dry milk followed by incubation overnight with anti-Ldb1 (1:2,000; see Supplementary Table 2 for additional antibody sources), anti-Isl1 (1:2,000), anti-Pdx1 (1:20,000), anti-Pax6 (1:1,000), anti-NeuroD1 (1:3,000; 3181-1; Epitomics), anti-Hnf1α (1:2,000, sc-6548X; Santa Cruz Biotechnology), and anti-MafA (1:2,000, A300 BL-1225; Bethyl Laboratories).

    Techniques: Expressing, In Situ Hybridization, Real-time Polymerase Chain Reaction, Isolation, Binding Assay, Immunohistochemistry

    Isl1-regulated MafA and Arx expression is greatly reduced in the E18.5 Ldb1 mutant pancreas. A : mRNA levels of islet-enriched transcription factors in E18.5 littermate control (blue bars) and Pax6-Cre ; Ldb1 F/F (red bars) pancreas ( n = 4–6). Littermate control mRNA level was set at onefold ± SEM. Immunostaining levels of β-cell MafA (red) ( B ) and α-cell Arx (red) ( C ) were greatly reduced in E18.5 Pax6 -Cre; Ldb1 F/F pancreata. Arrowheads in C mark Arx + glucagon + (white, top ) or Arx − glucagon + cells (yellow, bottom ), with some magnified hormone + cell clusters shown. D : ChIP analysis of Ldb1 binding to MafA Region 3 ( top ) as well as Arx Re1 and Re2 ( bottom ). The PEPCK promoter served as the negative background control. Dilute input as well as Ldb1- and IgG-enriched DNA were analyzed by PCR using βTC-3 and αTC-6 chromatin isolated from whole-cell extract (WCE) and/or nuclear extract (NE). H 2 O control serves as a negative control for the PCR. E : Binding between endogenous Ldb1 and Isl1 were found in coimmunoprecipitation experiments using βTC-3 nuclear extracts, whereas Ldb1 and Isl1 did not bind to Pdx1, NeuroD1, Hnf1α, MafA, or Pax6. Diluted βTC-3 nuclear extract served as input positive control (1%), and immunoprecipitation (IP) results were compared with species-matched IgG treatments. F : Dominant-negative acting Ldb1ΔN significantly reduced MafA region 3-driven reporter expression in βTC-3 cells. Data are presented as mean fold reporter activity, with the empty pFox-Luc + CMV cotransfection set at onefold ± SEM; n = 3. * P

    Journal: Diabetes

    Article Title: Islet ?-, ?-, and ?-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor

    doi: 10.2337/db12-0952

    Figure Lengend Snippet: Isl1-regulated MafA and Arx expression is greatly reduced in the E18.5 Ldb1 mutant pancreas. A : mRNA levels of islet-enriched transcription factors in E18.5 littermate control (blue bars) and Pax6-Cre ; Ldb1 F/F (red bars) pancreas ( n = 4–6). Littermate control mRNA level was set at onefold ± SEM. Immunostaining levels of β-cell MafA (red) ( B ) and α-cell Arx (red) ( C ) were greatly reduced in E18.5 Pax6 -Cre; Ldb1 F/F pancreata. Arrowheads in C mark Arx + glucagon + (white, top ) or Arx − glucagon + cells (yellow, bottom ), with some magnified hormone + cell clusters shown. D : ChIP analysis of Ldb1 binding to MafA Region 3 ( top ) as well as Arx Re1 and Re2 ( bottom ). The PEPCK promoter served as the negative background control. Dilute input as well as Ldb1- and IgG-enriched DNA were analyzed by PCR using βTC-3 and αTC-6 chromatin isolated from whole-cell extract (WCE) and/or nuclear extract (NE). H 2 O control serves as a negative control for the PCR. E : Binding between endogenous Ldb1 and Isl1 were found in coimmunoprecipitation experiments using βTC-3 nuclear extracts, whereas Ldb1 and Isl1 did not bind to Pdx1, NeuroD1, Hnf1α, MafA, or Pax6. Diluted βTC-3 nuclear extract served as input positive control (1%), and immunoprecipitation (IP) results were compared with species-matched IgG treatments. F : Dominant-negative acting Ldb1ΔN significantly reduced MafA region 3-driven reporter expression in βTC-3 cells. Data are presented as mean fold reporter activity, with the empty pFox-Luc + CMV cotransfection set at onefold ± SEM; n = 3. * P

    Article Snippet: The membrane was blocked in PBS/Tween supplemented with 5% nonfat dry milk followed by incubation overnight with anti-Ldb1 (1:2,000; see Supplementary Table 2 for additional antibody sources), anti-Isl1 (1:2,000), anti-Pdx1 (1:20,000), anti-Pax6 (1:1,000), anti-NeuroD1 (1:3,000; 3181-1; Epitomics), anti-Hnf1α (1:2,000, sc-6548X; Santa Cruz Biotechnology), and anti-MafA (1:2,000, A300 BL-1225; Bethyl Laboratories).

    Techniques: Expressing, Mutagenesis, Immunostaining, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Isolation, Negative Control, Positive Control, Immunoprecipitation, Dominant Negative Mutation, Activity Assay, Cotransfection