Gel mobility shift reactions using whole cell extracts from cell lines and patient samples prepared on a small scale by high-salt extraction demonstrate c-jun/AP-1 binding activity. (A) Extracts from the Ph⁺ model cell lines K562, Bcr-Abl.A54 (the murine cell line expressing p210 BCR-ABL), and patient sample no. 2271 were placed in gel mobility shift reactions with commercial oligonucleotide for the 7-base collagenase TRE (left) or a CTF/NF-1 oligonucleotide, which was used to control for protein loading by assay of a constitutively abundant transcription factor. Note relative abundance of c-jun/AP-1 binding activity. (B) Increased c-jun/AP-1 binding activity present in the patient sample is specific. On the left, another gel mobility shift reaction was performed with the 7-base collagenase TRE(AP-1) oligonucleotide including an extract, also prepared by the same high-salt extraction protocol, from a cell line (T47 breast cancer) known to be deficient in c-jun/AP-1 activity (HL-60 lysates could not be prepared by this protocol because of extensive autodigestion that could not be effectively inhibited). On the right, specificity of binding by extract from patient sample no. 2271, as well as from lysate of patient no. 2306 (known to harbor constitutive JNK activity), are shown to be specific because of cold-competition of their band-shift complexes formed with the collagenase TRE oligonucleotide by nonradioactive collagenase TRE at 100-fold excess. Note that the intensity of band-shift complexes formed is proportionately reduced after decay of the probe subsequent to labeling.