FIGURE 2.

No evidence for reversal of ARD hydroxylation over an extended period as determined by SILAC-chase methodology. A, synopsis of SILAC-chase methodology. HEK293 cells stably transfected with Rabankyrin-5 were grown in media containing heavy isotopes (Heavy media; Lys⁶/Arg⁶) for 5 days to achieve >90% incorporation of the mass label. Cells were subsequently “chased” in normal media (Light media; Lys⁰/Arg⁰), supplemented with 1 mm DMOG, to inhibit FIH-mediated hydroxylation. Following inhibition of FIH, the extent of hydroxylation on the pre-existing heavy labeled material was monitored over 36 h by LC-MS to determine the proportion of material that was hydroxylated over time. B, quantitation of heavy hydroxylation at Asn⁴⁸⁵ in Rabankyrin-5 over 36 h following addition of light media and DMOG. Data points were derived from extracted ion chromatograms of m/z 560.53 and 564.53 corresponding to heavy labeled forms of the unhydroxylated and hydroxylated tryptic Asn⁴⁸⁵ Rabankyrin-5 peptide, AAGAGNEAAALFLATNGAHVNHR ([M + 4H]⁴⁺) and expressed as percentage hydroxylation (Heavy Ox %). The overall percentage of heavy label incorporation, calculated from the sum of unhydroxylated and hydroxylated Asn⁴⁸⁵ heavy peptides over light forms of the peptide are depicted (black triangle), showing the expected removal of the heavy label over time. Raw data (extracted ion chromatograms for Asn⁴⁸⁵ peptides) are presented in supplemental Fig. S25 along with control data confirming the efficacy of DMOG treatment on the newly synthesized light material (supplemental Fig. S26).