Fig. 2: Proof-of-principle SIESTA experiment revealed known TXNRD1 substrates and suggested novel candidates.
From: System-wide identification and prioritization of enzyme substrates by thermal analysis
a Scatterplot of protein Tm differences upon addition of NADPH in lysate. Known proteins from UniProt are shown in red (n = 2 independent biological replicates; two-sided Student t-test; no adjustment for multiple comparisons was performed). b Representative stabilization of NADPH binding protein IDH1. c Scatterplot of Tm differences reveals the Tm shifts occurring only after simultaneous TXNRD1 + NADPH addition; these shifts are thus likely due to enzymatic modifications (yellow shaded area, known and putative substrates are shown as green circles). d Potential substrates (green circles) are mostly located close to the negative reference point (blue star) in an OPLS-DA model contrasting the TXNRD1 + NADPH Tm against the single treatments. Proteins shown in green are those identified as substrates in c. e Representative melting curves of GPX1, PRDX2, GULP1, and GSTO2 are shown. f Reduction of cysteines in the substrate proteins by incubation with TXNRD1 + NADPH (n = 3 independent biological replicates, one-sided Student t-test), measured by sequential iodoTMT labeling (Center line—median; box limits contain 50% of data; upper and lower quartiles, 75 and 25%; maximum—greatest value excluding outliers; minimum—least value excluding outliers; outliers—more than 1.5 times of the upper and lower quartiles) (NADPH nicotinamide adenine dinucleotide phosphate, Tm melting temperature, TXNRD1 thioredoxin reductase 1). Source data are provided as a Source Data file.
