FIGURE 4.

Fibronectin is a novel target of JMJD6-mediated lysyl hydroxylation and glycosylation. (A) Schematic of FN protein indicating sites involved in mediating FN–FN association during fibrillogenesis (black arrows). Table of FN protein sequences within the afore-indicated sites with a 100% probability of having lysine residues that are hydroxylated (red arrows). Sequences and probabilities were determined using the RF Hydroxysite prediction software. “***” represents the degree of highest probability of lysyl hydroxylation (100%). (B) (left panel) Western blot for FN following exposure of TC pMSCs to 10 μM minoxidil (Mxdl) for 48 h. n = 2 separate pMSC isolations. (B) (right panel) Western blot for FN in the insoluble matrix-associated protein fraction in TC pMSCs exposed to Mxdl. Representative of two separate pMSC isolations. (C) Western blot for FN and concanavalin A (Con A) following immunoprecipitation of FN upon Mxdl exposure (top panel) and JMJD6 siRNA knockdown (siJMJD6, bottom panel) in TC pMSCs (n = 3 separate pMSC isolations). Anti-rabbit IgG served as a negative control. (D) Representative mass spectra for FN peptide sequence 2 indicating the original peptide mass (top panel) and a 16-mass unit shift upon incubation with recombinant JMJD6 enzyme (200 ng) in the presence of ferrous iron (Fe²⁺; bottom panel).