annealing temperature, amplicon size and number of cycles, are listed in Table S1. For Western blot analysis, total protein content was determined according to Bradford [51]. Cell lysates were separated by SDS- polyacrylamide gel electrophoresis (5 mg total protein per lane) on a 10% separating gel and transferred onto a PVDF-membrane (Millipore) using a TE 77 semi-dry transfer unit (Amersham Bioscience). Membranes were then blocked overnight at 4uC, using 5% of milk powder in TBST (0.15 M NaCl, 10 mM Tris, 0.05% (v/v) Tween 20, pH 8.0). Proteins of interest were visualized using the antibodies described above and the enhanced chemoluminescence substrate Plus-ECL (PerkinElmer). Supporting Information Figure S1 Deletion and substitution-mutant analysis of the LKB1 promoter. (A) Comparison of luciferase activity of transiently transfected C33a cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-regulatory elements (left). The positions of the potential CCAAT boxes I–III as well as the forkhead box are indicated. Luciferase activity of all deletion constructs (relative light units normalized against renilla luciferase activity) is expressed as the percentage of the signal obtained with the plasmid containing the LKB1 promoter region downstream of nucleotide 2345 (LKB1 Pro III). (B) Luciferase activity of transiently transfected C33a cells with LKB1 promoter 10 bp substitution mutants reveals a critical role of four cis-acting elements regulating LKB1 transcription (dark grey bars). Activity of substitution mutants (relative light units normalized against renilla luciferase activity) is expressed as the percentage of the References 1. Jeghers H, Mc Kusick V, Katz KH (1949) Generalized intestinal polyposis and melanin spots of the oral mucosa, lips and digits; a syndrome of diagnostic significance. N Engl J Med 241: 1031–1036. 2. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, et al. 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