Fig. 1.

Gpx1 undergoes palmitoylation predominantly at Cysteine-76 and Cysteine-113.

(A–C) Gpx1, expressed endogenously in mouse retina (A), HEK293T cells (B), or HUVECs (C), was analyzed for protein palmitoylation using the ABE assay. (HA refers to hydroxylamine, HA + for treatment with HA, HA-for treatment without HA). (D–G) Cultured HUVECs were treated with either PalmB (D) or 2-BP (E), and lysates were evaluated for the level of Gpx1 palmitoylation using the ABE assay. The palmitoylation levels were quantified (F–G). (H) Cysteine conservation was analyzed by aligning the protein sequences of Gpx1 from different species. (I) Purified Gpx1 was probed using Mass-Spectrometry, and a mass-shift of 238 Da linked to cysteine was observed as a hallmark of palmitoylation. (J–K) Gpx1 constructs with point mutations (cysteine to serine) were expressed in HUVECs and subjected to evaluation of palmitoylation levels using the ABE assay (J), and the levels were quantified (K). (L) The palmitoylation modification sites of Gpx1 were analyzed by mPEG labeling assay in HUVECs. (M) HUVECs were metabolically labeled with 17-ODYA and the lysates were reacted with biotin-azide and enriched with streptavidin-agarose beads, hydroxylamine (HA) was used to hydrolyze thioester linkage and remove 17-ODYA labeling. Blots shown are representative, and data represents the mean ± S.E.M. from three biological replicates. Statistical significance is denoted as ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, determined by student t-test or one-way ANOVA followed by Tukey's post hoc test.