Gel mobility shift assays for protein extracts from atherosclerotic lesions. Intima and media (I/M) from chow-fed rabbits and media (M) and atherosclerotic intimal lesions (AS) from cholesterol-fed rabbits (16-week diet) were dissected and protein extracts were prepared as described in the Materials and Methods. a: Gel mobility shift assay performed using protein extracts (20 μg protein per lane) from I/M of control animals, atherosclerotic lesions (AS), or media (M). Arrowhead indicates protein-DNA-binding complexes; NS, nonspecific binding. b: Extracts obtained from atherosclerotic lesions were incubated with a radiolabeled oligonucleotide containing a HSF-binding site (HSE) with no addition (−), in the presence of unlabeled NF-κB, or unlabeled HSE, oligonucleotides (50:1). c: Protein extracts obtained from the media (M) or atherosclerotic lesions (AS) were incubated with a radiolabeled oligonucleotide containing a HSF-binding site in the presence of antibodies (1:20, Ab) specific to HSF1. Supershifted DNA-binding complexes indicative of HSF1 protein-containing complexes are indicated by the arrow. Each lane on the blot (a) represents an individual animal.