(a) Total plasma N-acyl amino acid levels from AAV-GFP (black) or AAV-PM20D1 (gray) transduced mice. Plasma was harvested after a one-week transduction period using a dose of 10e10 GC/mouse (intravenously).
(b,c) C20:4-Gly hydrolysis activity (blue traces), protein concentrations (orange traces), and Western blots (bottom panels) of fractionated mouse plasma one week after transduction by AAV-PM20D1-flag (a) or AAV-GFP (b).
(d,e) C20:4-Gly hydrolysis (c) and C18:1-Phe synthesis (d) activity of fractionated mouse plasma from either WT (blue trace) or PM20D1-KO (orange trace) mice.
(f) C20:4-Gly hydrolysis activity (blue traces), protein concentrations (orange traces), and Western blots (bottom panels) of fractionated pooled human plasma.
For (b-e), plasma from five mice per group was pooled together and separated by fast protein liquid chromatography-size exclusion (FPLC-SEC). For (a), (d), and (e), Student’s two-tailed t-test for AAV-GFP versus AAV-PM20D1 or WT versus PM20D1-KO across the indicated fractions was used to determine statistical significance. Data are shown as means ± SEM.