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IP-FCM detects the specific inhibition of CD25:IL-2 interaction by the first-generation SMIPPI, Ro-26-4550.

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posted on 19.02.2013, 22:56 authored by Stephen E. P. Smith, Anya T. Bida, Tessa R. Davis, Hugues Sicotte, Steven E. Patterson, Diana Gil, Adam G. Schrum

(A) Recombinant human CD25 (rhCD25) was immunoprecipitated with anti-CD25 antibody-conjugated CML beads. Recombinant human IL-2 (1000 U/ml) and one of several small-molecules were mixed with the beads for one hour, followed by probing with an anti-IL-2 antibody. Inhibition of the IL2:CD25 interaction was observed only upon addition of 10 mM R0-26-4550 (R26, blue shaded histogram). (B) Median fluorescence intensity from three experiments shows R26 significantly reduced IL-2:CD25 binding compared to either vehicle (DMSO) or PP2 negative controls (ANOVA: F3,10 = 10.82, p = 0.0051; **p<0.01, *p,0.05) (C) Endogenous CD25 was immunoprecipitated from lysates from PMA/Ionomycin-stimulated human PBMCs, and analyzed as in (A) using 10,000 U/ml IL-2. (D) Median fluorescence intensity from four experiments including (C) shows R26 significantly reduced IL-2:CD25 binding compared to either vehicle (DMSO) or PP2 negative controls (ANOVA: F3,15 = 10.07, p = 0.0013; **p<0.01) (E) Experiments for (C–D) included IL-2 and CD25 ELISAs that were performed. Compared to negative controls, R26 did not change the amount of IL2 or CD25 detected (N.S, not significant by Student’s T-test, p>>0.05), indicating that the difference in (C–D) was due altered IL-2:CD25 interaction.

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