Effect of AG-348 on protein phosphatase, PTP1B. (A-B) Ex vivo effect of AG-348 and R406 on Tyr-p-bd3. HbSS RBCs were treated with DMSO (Con) or different concentrations (3, 10, and 30 μM) of R406, AG-348, or a combination of R406 and AG-348 for 6 hours, and the level of Tyr-p-bd3 was analyzed by western blotting and quantified by densitometry analysis. The mean reductions on Tyr-p-bd3 of RBCs treated with 10- and 30-μM R406, AG-348, or a combination compared with vehicle control were 15% and 36%, 6% and 31%, 39% and 66%, respectively. ∗∗P ˂ .01; ∗∗∗P ˂ .001. (C-D) Effect of AG-348 on RBC PTP1B. HbSS RBCs were treated with DMSO (Con) or 30-μM AG-348 in DMSO for different times (1, 2, 4, and 6 hours), and the level of membrane-associated intact PTP1B was analyzed by western blotting and quantified by densitometry analysis. The mean increase on PTP1B of RBCs treated with 30-μM AG-348 for 4 and 6 hours were 25% and 43%, respectively. ∗P ˂ .05. (E) Immunoprecipitation with anti-PTP1B antibody clearly showed interaction between PTP1B and band 3. (F-G) HbSS RBCs were treated with DMSO (Con) or different concentrations (1, 3, 10, and 30 μM) of AG-348 for 8 hours, and the interaction between PTP1B and band 3 was measured by immunoprecipitation and quantified by densitometry analysis. The mean increase on the interaction between PTP1B and band 3 of RBC treated with 1-, 3-, 10-, and 30-μM AG-348 were 31%, 35%, 70%, and 121%, respectively. ∗P ˂ .05. Three biological replicates of HbSS donors were used in all ex vivo assays. IgG, immunoglobulin G.