For protein preparation, wild-type PTPROt or the C325S mutant, each carrying a C-terminal FLAG tag, was expressed in HEK293 cells and purified by immunoprecipitation. Inactive, phosphorylated, HA-tagged wild-type PTPROt was prepared similarly from transfected HEK293 cells that had been treated with 0.5 mM sodium pervanadate for 5 min immediately before lysis. Lysis and precipitation buffers did not contain sodium pervanadate. After immunoprecipitation, beads carrying the FLAG-tagged proteins were washed three times with HNTG buffer [20 mM Hepes (pH 7.3), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol] and twice with elution buffer [20 mM tris (pH 7.5), 100 mM NaCl, and 0.5 mM EDTA]. FLAG-tagged proteins were eluted from the beads by incubation for 10 min at 37°C in 3XFLAG peptide (equal volume of 1 mg/ml; APExBIO in elution buffer). Elution was repeated and both eluates were combined. Inactive, HA-tagged, phosphorylated PTPROt was not eluted. Protein purity and amount were assessed relative to a BSA standard by SDS-PAGE.

For the PTPROt trans-dephosphorylation activity assay, equal amounts of beads carrying inactive, HA-tagged, phosphorylated PTPROt, prepared as described above, were suspended in 100 μl of phosphatase assay buffer [50 mM MES, 0.5 mM dithiothreitol, and BSA (0.5 mg/ml)] along with 200 ng of FLAG-tagged wild-type or C325S PTPROt eluted protein, and incubated at 32°C for 30 or 60 min. Sodium pervanadate (5 mM) was added to some reactions. The beads carrying HA-PTPROt were then washed, subjected to SDS-PAGE, and analyzed by protein blotting for pTyr and HA.

To assay the activity of mutant PTPROt molecules, FLAG-tagged wild-type PTPROt and its mutant forms (C325S, R331M, or Y399F PTPROt) were separately expressed in HEK293 cells. Cells were lysed in buffer A, and lysates were verified by protein blotting to contain similar amounts of the PTPROt proteins. Tyrosine phosphatase activity present in the lysates was measured in 96-well plate format, each well containing 25 or 50 μg of lysate protein in 200 μl of phosphatase assay buffer [50 mM MES, BSA (0.5 mg/ml), 0.5 mM dithiothreitol, and 10 mM PNPP (pH 7.0)]. Some reactions contained 0.5 mM sodium pervanadate to identify PTP activity, which is inhibited by pervanadate. Activity was monitored as the increase in absorption at 405 nm at 30°C for 1 hour and was linear with respect to time and amount of lysate. Activity of endogenous tyrosine phosphatases was determined in lysates of mock-transfected cells and was subtracted from activity measured in PTPROt-expressing cells.

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