Figure 6. A putative secreted Klotho protein is not detected in HK-2 cell supernatant.

(A) Western blot for Klotho on HK-2 cell lysate, 48 hour–conditioned supernatant, and recombinant human (Rh) Klotho. Note the 130 kDa bands corresponding to full-length Klotho in HK-2 cells and in supernatant, similar to 130 kDa positive control recombinant Klotho. While a 70 kDa KL1 band was detected in the positive control, no such band was detected in HK-2 cell supernatant. Note that the relative densities of the recombinant protein and KL1 fragment are unrelated to the expected secondary cleavage of 130 kDa soluble Klotho generated by HK-2 cells. Smaller bands were determined to correspond to albumin and a likely immunoglobulin chain, as detailed in Supplemental Figure 3. (B) Ponceau S protein staining showing a prominent band at 65 kDa in HK-2 supernatant, corresponding to albumin, which is likely residual BSA from FCS. This band is further characterized in Supplemental Figure 3.