Figure 5.

Sialidase treatment increased the activity of heavily sialylated CHO-sKlotho by 3-fold but had little effect on the less-sialylated HEK-sKlotho in the FGF23 ERK1/2 activation assay. A, CHO- or HEK-sKlotho proteins (0.5 mg each) were treated or mock-treated with sialidase (500 units; New England Biolabs) overnight at room temperature in G1 buffer, repurified through a nickel-nitrilotriacetic acid column, buffer-exchanged, and concentrated at 0.4 mg/ml in PBS buffer. The protein samples were analyzed by SDS-PAGE or IEF gel. B, sialidase-treated or mock-treated sKlotho proteins were measured in the FGF23 ERK1/2 activation assay as described under “Experimental procedures.” The error bars at each data point were calculated from a triplicated experiment.