Northern and band-shift analyses of DC3F and A3 Chinese hamster lung fibroblasts. (A) Northern blot of total RNA probed with mMsh3 cDNA probe. The middle panels show an overexposure of the top panels. The positions of the 28S and 18S rRNA bands are shown. The size of the msh3 transcript is in agreement with a previous study (39). The lower panels represent total RNA probed with human β-actin cDNA, to verify equal loading. (B) Band-shift assays in which extract proteins were incubated with labeled DNA oligonucleotide duplexes containing a mismatch (G⋅T), three unpaired nucleotides (TTT), or no mismatch (A⋅T). The reaction mixtures were electrophoresed in 6% polyacrylamide gels; the positions of the DNA duplexes in complex with MutSα (α) and MutSβ (β) are shown, and other unlabeled bands represent nonspecific protein⋅DNA complexes. The specificity of MutSα and MutSβ binding was verified by competition assays using a 40-fold molar excess of unlabeled duplex (data not shown), and results were consistent with a previous study (45).