Journal: Diabetes

Article Title: Both Polymorphic Variable Number of Tandem Repeats and Autoimmune Regulator Modulate Differential Expression of Insulin in Human Thymic Epithelial Cells

doi: 10.2337/db10-0255

Figure Lengend Snippet: VNTR responded sensitively and differentially to AIRE in a human thymic epithelial cell line. A : The VNTR responded to AIRE in a hTEC line. A class I VNTR (PQ) and a class III VNTR reporter construct (50 ng) were cotransfected with AIRE expression vector or control vector β-gal DNA into a hTEC line. The amount of AIRE expression vector added to each sample of a 24-well plate format is indicated. B : Western blot analysis was performed to confirm that the increasing expression of AIRE protein corresponded to the amounts of added expression vector. Equal loading of each sample was confirmed by incubation with an anti-α-actin antibody.

Article Snippet: In addition, a second rabbit antibody recognizes the epitope 246-545 amino acids in AIRE protein (Santa Cruz) was used to detect the immunoprecipitated AIRE proteins.

Techniques: Construct, Expressing, Plasmid Preparation, Control, Western Blot, Incubation

Journal: Diabetes

Article Title: Both Polymorphic Variable Number of Tandem Repeats and Autoimmune Regulator Modulate Differential Expression of Insulin in Human Thymic Epithelial Cells

doi: 10.2337/db10-0255

Figure Lengend Snippet: AIRE regulates the insulin promoter activity mediated through the INS-VNTR element. A and B : Insulin basal promoter, VNTR-class I-INS-basal and VNTR-class III-INS-basal vectors demonstrated differential promoter activities in both hTEC and rat primary TEC in response to AIRE. C : DNA probes derived from insulin basal promoter (0.4 kb), VNTR-class I-INS-basal (2.0 kb), and VNTR-class III-INS-basal (3.5 kb) were radiolabeled with T4 polynucleotide kinase and γ- 32 P-ATP as shown in the input lanes. An Ad-AIRE transduced hTEC total lysate (100 μg) was mixed with 50,000 cpm of each radiolabeled DNA probe for 6 h at room temperature and subsequently added 1 μg of goat anti-AIRE antibody for an overnight incubation. The immune complex was precipitated by salmon sperm DNA-treated protein G-agarose beads, washed, and eluted from beads for separation. Normal goat IgG was used as a negative control. The immunoprecipitated AIRE complexes were verified using an anti-AIRE antibody. D : VNTR linked to a heterologous promoter failed to respond to AIRE. The insulin basal promoter was replaced by a SV40 promoter in a reporter construct. Transfection of the construct lacking the insulin basal promoter with the AIRE expression vector in human TEC was measured for luciferase reporter activity. E : Addition of a SV40 enhancer to the construct greatly enhanced SV40 promoter activity, but the class I or III VNTR had no effect on the promoter/enhancer combination. Transfection efficiency was normalized with the null- Renilla luciferase reporter vector.

Article Snippet: In addition, a second rabbit antibody recognizes the epitope 246-545 amino acids in AIRE protein (Santa Cruz) was used to detect the immunoprecipitated AIRE proteins.

Techniques: Activity Assay, Derivative Assay, Incubation, Negative Control, Immunoprecipitation, Construct, Transfection, Expressing, Plasmid Preparation, Luciferase