Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Imaging NF-κB activity in a murine model of early stage diabetes

doi: 10.1096/fj.201801147R

Figure Lengend Snippet: A. Molecular model of the near-infrared ODN duplex probe (NIR-ODND) with a single NF-κB consensus binding site carrying a Cy5.5 fluorophore on one of the ODN strands. B-electrophoretic mobility (4–20% polyacrylamide gradient /TBE) of the NIR-ODND and single ODNs. ODND was labeled with either Cy5.5 (lane 3, red) or 800CW (lane 4, green). The migration of single strand NIR-fluorophore labeled ODNs serve as controls and are shown in lanes 1 and 2. C-electrophoretic mobility shift assay (EMSA, 10% PAGE): quantitative analysis of shifts using Cy5.5-labeled ODND (fluorescence emission – 700 nm) as a probe for activated NF-κB in pancreatic extracts collected on day 8. NIR-ODND was incubated in the presence of: lane 1- none; lane 2- HeLa nuclear extract (positive control); lane 3- islet nuclear extract-control; lane 4 - islet nuclear extract-LD-STZ; lane 5- islet cytoplasmic extract-control; lane 6- islet cytoplasmic extract-LD-STZ. The arrowhead points to the shifted ODN bands. D-quantification of fluorescence intensity of shifted bands due to NIR-ODND banding to the components of islet extracts. E-electrophoretic mobility shift assay for testing the specificity of NIR-ODND binding: lane 1- Cy5.5-labeled ODND, lane 2- HeLa nuclear extract; lane 3 – HeLa and excess of non-labeled ODND; lane 4: inactive HeLa extract; lane 5- LD-STZ treated islet extract; lane 6- LD-STZ treated islet extract and excess of non-labeled ODND; lane 7- inactive LD-STZ treated islet extract. For testing band supershift in the presence of anti- NF-κB p65 antibody Cy3-labeled ODND was analyzed on 7.5% PAGE gels: lane 8- ODND only, lane 9 - LD-STZ treated islet extract; lane 10 - LD-STZ treated islet extract and anti- NF-κB p65 antibody.

Article Snippet: Images were captured using Nikon TE2000-U inverted microscope equipped with a standard Cy5.5 set (XF141–2, Omega optical).

Techniques: Binding Assay, Labeling, Migration, Electrophoretic Mobility Shift Assay, Fluorescence, Incubation, Positive Control