Journal: Aging (Albany NY)

Article Title: Five ETS family members, ELF-1, ETV-4, ETV-3L, ETS-1, and ETS-2 upregulate human leukocyte-associated immunoglobulin-like receptor-1 gene basic promoter activity

doi: 10.18632/aging.101475

Figure Lengend Snippet: Binding of ETS-2 to the human LAIR-1 gene basic promoter. ( A ) CHIP assays. Chromatins from Jurkat (Left) and COC1 cells (Right) were prepared. Shown are the PCR products from: Lane 1, Chromatin before immunoprecipitation (input chromatin); Lane 2, Chromatin incubated with rabbit anti-ETS-2 antibodies; Lane 3, Chromatin incubated with negative control (normal rabbit IgG). ( B , C and D ) Electrophoretic mobility shift assays (EMSA). Nuclear extracts from Hela cells were subjected to EMSA. The use of biotin-labeled oligonucleotide probes A , B and C carrying the ETS binding site A , B and C , respectively, allowed assessment of the specificity of the protein-DNA binding. Competitors were unlabeled oligonucleotide probes, supplied at 20-fold and 100-fold concentrations. Specific binding: protein-DNA complexes of proteins and the probes. NSB: nonspecific binding. Free probe: free biotin-labeled oligonucleotide probes that did not bind to the proteins. The experiments were repeated at least three times.

Article Snippet: EMSA assays were performed using the Complete Non-radioactive EMSA kit (Viagene Biotech Co., Ningbo, China), according to the manufacturer’s instructions and a biotin end-labeled DNA duplex of sequence containing ETS-2 putative binding site A (5’- TCT CAC TAG TGG GAA GGG TGG AAA GAA CAG-3’; 5’-CTG TTC TTT CCA CCC TTC CCA CTA GTG AGA-3’ ), site B (5’-GGA CAG AAA GCT CTT CCT CTT GTG TGA GGC-5’; 5’-GCC TCA CAC AAG AGG AAG AGC TTT CTG TCC-3’) or site C (5’-CGG GCA GCA CAT CCT GTC TGC TTG TGT c-3’; 5’-GAC ACA AGC AGA CAG GAT GTG CTG CCC-3’).

Techniques: Binding Assay, Immunoprecipitation, Incubation, Negative Control, Electrophoretic Mobility Shift Assay, Labeling