Recombinant HA-NFATp is phosphorylated and displays regulated nuclear association in vitro. (A) HA-NFATp is phosphorylated in its N-terminal region and can be dephosphorylated by calcineurin in vitro. Full length NFATp (lanes 1 and 2) and three deletion mutants (lanes 3-8) were expressed with N-terminal HA-tags using recombinant baculoviruses. All four proteins were purified by anti-HA affinity chromatography and eluted with HA-epitope peptide. The proteins were individually incubated with calcineurin and calmodulin (CaM, even lanes) or mock treated in reactions lacking calcineurin and calmodulin (odd lanes). Proteins were resolved by SDS-PAGE and visualized by silver staining. The position of the calcineurin in the SDS gel is indicated. Note the shift in migration of HA-NFATp(1-921) and HA-NFATp(1-722) upon treatment with phosphatase. (B) HA-NFATp associates with nuclei in vitro only after treatment with calcineurin. HA-NFATp that was pre-treated with calcineurin and CaM (lanes 5 and 6) or mock treated (lanes 3 and 4) was mixed with HeLa nuclei. A control reaction was performed without added HA-NFATp (lanes 1 and 2). After incubation, the nuclei were pelleted by centrifugation and washed. Protein in the pelleted nuclei fractions (P) or supernatant fractions (S) were resolved by SDS-PAGE and analyzed by protein immunoblotting with anti-HA antibody. The positions of phosphorylated HA-NFATp and dephosphorylated HA-NFATp are indicated. The position of a non-specific protein in HeLa nuclei that is recognized by the anti-HA antibody is indicated with an asterisk.