Identification of PP2A as the phosphatase responsible for the dephosphorylation of hyperphosphorylated paratarg-7. (A) Inhibition of phosphatases: LCLs derived from a healthy donor were cultured in the presence of inhibitors at indicated concentrations. Lysates were analyzed by IEF and stained with anti-paratarg-7. LCL lysates derived from a patient carrying hyperphosphorylated paratarg-7 (lane b) or from a healthy donor carrying wild-type paratarg-7 (lane a) and HEK293 cells (expressing wild-type paratarg-7, lane c) were used as controls. Incubation with serine/threonine phosphatase inhibitor cocktail 1 (Sigma-Aldrich; dilution 1:1000; lane d) and ocadaic acid (lane f, 500nM; lane g, 10nM; lane h, 0.1nM) inhibited the dephosphorylation resulting in the appearance of hyperphosphorylated paratarg-7. NIPP1 (10mM; lane e) had no effect. NIPP1 is a specific inhibitor of protein phosphatase 1 (PP1); ocadaic acid at 10nM inhibits both PP1 and PP2A, while at concentrations equal or below 0.1nM it inhibits only PP2A. (B) Analysis of PP2A subunit C derived from MM/MGUS patients and healthy donors: LCLs were subjected to IEF followed by immunodetection using PP2A subunit C Abs (1:1000; subpanel i) or pY307 specific Abs (1:1000; subpanel ii). The figure shows that in carriers of wild-type paratarg-7 (lanes a-b) the bottom band (representing the nonphosphorylated subunit) is dominant, while in patients carrying hyperphosphorylated paratarg-7 (lanes c-e) the upper band (representing the phosphorylated subunit) is dominant. Subpanel ii shows that the top band represents subunit C phosphorylated on Y307. Samples as in panel A.