FIG. 2.

Generation and characterization of recombinant H1 protein phosphatase activity. (A) Schematic representation of sequence differences in VACV and VARV H1. The C110 residue that is required for catalytic activity is also indicated. (B) SDS-PAGE (4–12% gradient) and Coomassie blue staining of total lysates (L) or purified proteins (P) from bacteria expressing VACV H1 (1), VARV H1 (2), VACV C110S H1 (3), or VARV C110S H1 (4). (C) Immunoblot analysis of purified recombinant proteins using mouse monoclonal antibody directed against the Xpress tag. P1: VACVH1; P2: VARVH1; P3: VACVC110S H1; P4: VARVC110S H1. (D) Hydrolysis of p-nitrophenyl phosphate by H1 proteins. Hydrolysis is measured as increase in optical density at 405 nm. Data are presented as the mean ± SE of triplicate determinations and are representative of three experiments. (E) Calculation of enzyme activity based on activity at 30 min observed in (D). One unit is the amount of phosphatase required to release 1 nmol phosphate from pNPP in 1 min under the tested assay conditions. Data are presented as mean ± SE. (F) Determination of activity/μg of protein on the indicated peptide substrates (STAT1pY701 peptide, LDDPKRTGpYIKTELISVS; VACV A17pY203 peptide, PYTAGNKVDVDIPTFNSLNTDDpY). Activity was determined as the amount of dephosphorylation per pg protein and was averaged over values at several concentrations of enzyme. A17 phosphopeptides were detected by antiphosphotyrosine 4G10 mAb, and phospho-Stat1 levels were determined with antiphospho-Stat1. Results are representative of three experiments. (G) Stat1 or Stat3 was immunoprecipitated from NIH 3T3 cells stimulated, respectively, with IFN-γ and OSM. Precipitates were incubated with recombinant H1 proteins, and phosphorylation level was determined with immunoblot analysis.