(A) The α-klotho gene produces a single pass type I transmembrane protein with a short intracellular domain (11 amino acids), a transmembrane domain (21 amino acids) and a large extracellular domain (980 amino acids). The extracellular domain contains two homologous repeats termed KL1 and KL2. The transmembrane form is shed from the membrane by the action of ADAM10/17 (α-cut), within the sequence LGSGTLGRF to produce soluble klotho. The second cleavage site (β-cut), also carried out by ADAM10/17, is between the KL1 and KL2 domains, within the sequence PPLPENQPL. The secreted form of klotho is produced by alternative splicing of the mRNA with a premature stop codon in exon 3, producing only the KL1 domain with an additional 15 amino acids at the C-terminus; with the sequence changing from DTTLSQFTDLNVYLW to SQLTKPISSLTKPYH. The secreted form is directly secreted from the cell. The klotho-VS polymorphism is located in the KL1 domain and is therefore present in all three forms of α-klotho. (B,C) HEK293 cells were stably transfected with the cDNAs encoding full-length transmembrane human klotho, soluble klotho or secreted klotho. Lysates (B) and media (C) were collected and immunoblotted with antibody LS-C500248. In the cell lysate full-length klotho migrates at 135 kDa, while in the media it migrates at 130 kDa due to its proteolytic shedding. The soluble and secreted forms of klotho migrate at 130 and 70 kDa, respectively, and are detected in both the cell lysate and media. In the media secreted klotho appears as two forms likely due to proteolytic processing.