Fig. 3. HSF1—HSE complexes in the liver tissue of G. mirabilis visualized via electrophoretic mobility-shift assay (EMSA). A competition assay was run to determine HSE probe specificity. HSF-1 activation was visualized via EMSA, using a LightShift^TM chemiluminescent EMSA kit (Pierce^TM). After a 1 h heat shock at the indicated temperature, 3 µg of homogenized liver tissue were incubated with 15 pmol of biotinylated-DNA protein for 20 min at 18°C and then separated on a 5% non-denaturing polyacrylamide gel. Protein was blotted to nylon membrane, and HSF1—HSE complexes were visualized through a chemiluminescence reaction. Lane 1, liver tissue and biotinylated HSE probe only; lane 2, identical to lane 1 except for the addition of a 200 moll^-1 excess of an unlabelled non-competitor DNA probe (AP2 from Promega); lane 3, identical to lane 1 except for addition of a 200 moll^-1 excess of unlabelled HSE probe. Intensities of HSF1—HSE bands were determined by scanning densitometry (BioRad^TM Fluor S Multimager).