Fig. 4: Regulation of potential ADO substrate sequences in Dual-Fluorescence Oxygen Reporter (DFOR) assay.
From: N-terminal cysteine acetylation and oxidation patterns may define protein stability
a Sequences of proteins selected from in vitro screening for testing in cells; including RAP2.12, a substrate of related Plant Cysteine Oxidase (PCO) enzymes. Amino acids are colour coded according to chemical properties: basic (H/K/R) = red; acidic (D/E) = blue; aromatic (F/W) = orange; polar (S/T) = purple. “in vitro Activity” values are relative to RGS42-15 (%). b, c SH-SY5Y cell lines stably expressing MCX13GFP:P2A:mCherry were (b) treated for 20 h with 2,2-dipyridyl (200 µM) or DMSO vehicle control and (c) incubated for 24 h in normoxia or hypoxia (1% O2). GFP/mCherry ratio was calculated using fluorescence measurements and normalised to the GFP:mCherry ratio obtained in cells expressing the positive control construct RGS4(1-15)-GFP:P2A:mCherry. Higher GFP/mCherry ratio indicates stabilisation of eGFP protein; increased stability after dipyridyl or hypoxia treatment is consistent with reduced ADO activity. Error bars show SEM. Statistical significance determined with 2-way ANOVA with Holm-Sidak multiple comparisons post-hoc test: ns (p > 0.05); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001: ****p ≤ 0.0001. Data were collected across at least three biological repeats. Source data are provided as a Source Data file.
