Klotho gene expression and methylation. A) RT-PCR detection of klotho gene mRNA expression in GES-1 normal gastric epithelial cells and, MNK-45, AGS, and GC-7901 gastric cancer cells. B) Western blot of klotho protein expression. C) The relative klotho protein levels in B). D) Methylation of klotho gene promoter. The methylated (Me) and unmethylated (UM) klotho gene promoter fragments were amplified from bisulfite-treated genomic DNA by PCR. M: DNA ladder.