Fig 1: RedoxiFluor can quantify target-specific protein thiol redox state in relative percentage and molar terms. A. Click-PEG cannot detect PP2A redox state, as evidenced by the loss of signal in the “PEGylated” lanes (3–5) compared to lysates (lane 1) and the PEG-free clickable maleimide handle only control (lane 2). B. ALISA detected a significant difference (unpaired t-test, P = 0.0192, n = 3) between the 20 and 40% redox states. C. Protein A mode RedoxiFluor can accurately and reproducibly discern between different PP2A redox states ranging from 10 to 90% reversibly oxidised (n = 3 per standard, see methods). D. ELISA mode RedoxiFluor can accurately and reproducibly discern between different PP2A redox states ranging from 10 to 90% reversibly oxidised (n = 3 per standard, see methods). A separate PP2A ELISA mode standard experiment quantifying significant differences (unpaired t-tests, P = < 0.0001 in panels E–G, n = 6) between the 20 (n = 6) and 40% (n = 6) reversibly oxidised states in percentages (E) and picomoles of reduced (F) and reversibly oxidised (G) protein. All standards and samples were derived from Xenopus laevis lysates (see methods). Data are presented as the mean (M) and standard deviation (SD).
Fig 2: LPS increases PP2A-, PTP1B-, SHP1-, and CD45-specific reversible thiol oxidation. No significant difference (all unpaired t-tests and n = 6) in PTEN (P = 0.1871), SHP2 (P = 0.3054), and calcineurin (P = 0.2780) specific reversible thiol oxidation (i.e., percent oxidised protein) in unstimulated (control) and LPS-stimulated human monocytes as determined by array mode RedoxiFluor. A significant (all unpaired-tests and n = 6) LPS-induced increases in PP2A (P = 0.0181), SHP1 (P < 0.0001), PTP1B (P = 0.0483), and CD45 (P = 0.0018) specific reversible thiol oxidation occurred in LPS-stimulated human monocytes compared to unstimulated controls. Data are presented as the mean (M) and standard deviation (SD).
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