Activation of STAT molecules.

STAT molecules are constitutively activated in the HD cell lines L428 (A) and L540 (B). Analysis of the DNA-protein complexes formed between nuclear extracts from the L428 using radiolabeled oligonucleotides containing either the consensus or mutated STAT1/3 binding sites, by competition with corresponding unlabeled oligonucleotides. Supershift experiments were performed using antibodies directed against STAT1 and STAT3, and against STAT5 and PEA1 as negative controls. Nuclear extracts (5 μg) were analyzed in a gel retardation experiment using radiolabeled oligonucleotides. The nuclear extracts were incubated with the corresponding STAT1/3 (consensus) or STAT1/3-M1 (mutated GGAA to CTAG) oligonucleotide, the DNA-protein complexes were separated by electrophoresis using a native 4.5% polyacrylamide gel and visualized by autoradiography (Figure 1). Arrows show the position of STAT3 and STAT1 homodimers and heterodimers and supershifted bands; brackets and asterisks indicate the radiolabeled oligonucleotide used.