Fig. 2

CpG methylation of CRYAA promoter decreases the DNA-binding capacity of transcription factor SP-1. EMSA experiments show the specific binding of Sp1 to the binding sequence of the CRYAA promoter. Lane 1: biotin-labeled wild-type Sp1 probe incubated with HLE B-3 nuclear extracts. Sp1 binding is depicted by arrows on the left of the figures. Lane 2: excess unlabeled wild-type competitor (1:100) competes for binding with the labeled probe. Lane 3: unlabeled methylated competitor (1:100) does not compete with the labeled probe. Lane 4: labeled methylated probe incubates with nuclear extracts. No binding was shown. Lanes 5 and 6: excess unlabeled wild-type and methylated competitors (1:100) were added, respectively. No binding of Sp1 was shown. Lane 7 shows anti-Sp1 supershift. Anti-Sp1 antibody is added to nuclear extracts incubating with a wild-type probe. Lane 8 shows no binding of Sp1. Anti-Sp1 antibody is added to nuclear extracts incubating with a methylated probe