α-isl-1 antibody  (Developmental Studies Hybridoma Bank)

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Arx expression is elevated Pdx1PB-Isl-1-Myc mice. A, diagram representing the Pdx1PB-Isl-1-Myc transgenic construct, which contains the pdx1PB fragment of the pdx1 gene and the hsp68 minimal promoter. B, Western blot by α-Isl-1 and α-Myc analysis of total cell lysate from islets of 8-week-old control and Pdx1PB-Isl-1-Myc mice. The asterisk denotes a nonspecific band. C, Isl-1 mRNA levels in Pdx1PB-Isl-1-Myc and littermate control mice. Results are presented as mean ± S.E. *, p <0.05. D–G, immunohistochemical analysis for glucagon or insulin and Myc expression in 8-week-old Pdx1PB-Isl-1-Myc and control mice. H, Arx mRNA levels in Pdx1PB-Isl-1-Myc and control mice. Results are presented as mean ± S.E. *, p <0.05. I–L, costaining of Arx with glucagon or insulin in 8-week-old Pdx1PB-Isl-1-Myc and control mice. Insets show the magnified views of the outlined areas. The asterisk denotes autofluorescence from red blood cells.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Expressing, Transgenic Assay, Construct, Western Blot, Immunohistochemical staining

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: The Isl-1 binding sites in Re1 and Re2 are necessary for αTC1–6 cell activity. Sequence identity within Arx Re1 (A) and Arx Re2 (B) between mouse, human, and rat. Core Isl-1 binding site sequences are shown in bold, and the lines above demarcate the EMSA probe. Nucleotide mutated for luciferase reporter (C) and EMSA (Fig. 6C) assays are labeled in red. Nonconserved nucleotides are labeled in green. C, the activity of wild-type and Isl-1 binding site mutants (red) of pGL4.27-Re1 and pGL4.27-Re2 in αTC1–6 cells. The pRL-SV40 normalized data were presented as the mean ± S.E. *, p <0.05.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Binding Assay, Activity Assay, Sequencing, Luciferase, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Isl-1 binding sites in Re1 and Re2. A and B, as a screen of putative Isl-1 binding elements, the radiolabeled MafA-R3 Isl-1 site probe (17) was used in reactions with in vitro translated Isl-1-Myc and MafA-R3, Re1, or Re2 competitor oligonucleotides. Isl-1 and Myc-epitope antibodies were used to localize the Isl-1-Myc:MafA complex. Ab, antibody; IVT, in vitro translation; Neg, without Isl-1-Myc; Pos, with Isl-1-Myc. C, Re2–2, Re2–3, and Re2–6 probes were incubated with αTC1–6 nuclear extract. The specificity of Isl-1:Arx-Re2 binding was determined by Isl-1 antibody addition and competition with excess of unlabeled wild-type (WT) and Isl-1 binding site mutant (MT).

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Binding Assay, In Vitro, Incubation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Arx expression is elevated Pdx1PB-Isl-1-Myc mice. A, diagram representing the Pdx1PB-Isl-1-Myc transgenic construct, which contains the pdx1PB fragment of the pdx1 gene and the hsp68 minimal promoter. B, Western blot by α-Isl-1 and α-Myc analysis of total cell lysate from islets of 8-week-old control and Pdx1PB-Isl-1-Myc mice. The asterisk denotes a nonspecific band. C, Isl-1 mRNA levels in Pdx1PB-Isl-1-Myc and littermate control mice. Results are presented as mean ± S.E. *, p <0.05. D–G, immunohistochemical analysis for glucagon or insulin and Myc expression in 8-week-old Pdx1PB-Isl-1-Myc and control mice. H, Arx mRNA levels in Pdx1PB-Isl-1-Myc and control mice. Results are presented as mean ± S.E. *, p <0.05. I–L, costaining of Arx with glucagon or insulin in 8-week-old Pdx1PB-Isl-1-Myc and control mice. Insets show the magnified views of the outlined areas. The asterisk denotes autofluorescence from red blood cells.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Expressing, Transgenic Assay, Construct, Western Blot, Immunohistochemical staining

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Isl-1 binds to Re1 and Re2 of the mouse Arx gene. A, schematic diagram of the Arx locus illustrating the locations of Re1 (orange box, +5.6 to +6.1) and Re2 (green box, +23.6 to +24 kb). TSS, transcription start site; Ex, exon. B, the ChIP-Seq image depicts Isl-1 occupancy within Arx Re1 (chrX:89544959–89545500) and Re2 (chrX:89563002–89563440) in αTC1–6 cells. The conservation between mouse and other species within this region was shown using the University of California Santa Cruz genome browser. C and D, Re1 and Re2 binding to Isl-1 was readily detected in adult mouse islets and αTC1–6 cells by standard ChIP but to a lesser extent in βTC3 cells. Results were normalized to Isl-1 binding to the PEPCK promoter. Data are presented as the mean ± S.E. n = 3; p value <0.05.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Re1 and Re2 mediate Arx transcription in αTC1–6 cells. A, pGL4.27-Re1, pGL4.27-Re2, and pGL4.27-Re1/2 were transfected into αTC1–6 cells in the presence or absence of a Isl-1-Myc expression plasmid. B, Western blot analysis using Isl-1 and control α-tubulin antibodies demonstrates a ∼70% reduction of Isl-1 protein levels in Isl-1 shRNA lentivirus-treated αTC1–6 cells. C, endogenous Arx mRNA expression was down-regulated relative to HPRT in Isl-1 shRNA treated αTC1–6 cells. D, Isl-1 shRNA knockdown reduced pGL4.27-Re1 and pGL4.27-Re2 reporter activity in αTC1–6 cells. Results are presented as the mean ± S.E. *, p <0.05. pGL4.27-Arx activity was normalized to that of the pRL-SV40 Renilla luciferase in A and D.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, shRNA, Activity Assay, Luciferase