FIGURE 5.

Hydroxylation has minimal effect on the stability of overexpressed Rabankyrin-5. A, Rabankyrin-5 half-life was similar between cells treated with control siRNA (black triangle) or siRNA specific to FIH (gray circle) as demonstrated by a similar loss of heavy label signal during 48-h chase period in light media (Lys⁰/Arg⁰). B, confirmation that siRNA-mediated knockdown of FIH suppressed the hydroxylation of heavy labeled Rabankyrin-5 at Asn⁴⁸⁵ at the start (t = 0) and end (t = 48 h) of the chase period. Note, the apparent increase in heavy hydroxylation observed 48 h after label washout was noted previously (Fig. 4B); it most likely reflects the accrual of hydroxylation as the average age of the heavy labeled material increases. Raw data (extracted ion chromatograms for Asn⁴⁸⁵ peptides) are presented in supplemental Fig. S27. C, immunoblot demonstrating siRNA-mediated knockdown of FIH at the protein level through the duration of the chase period.