In vitro phosphatase assay and dimedone-labeling

Purified recombinant human PTP1B (1–321) was kindly provided by Dr N.K. Tonks (Cold Spring Harbor Laboratory, New York). PTP1B (0.5 μg ml⁻¹) was pre-incubated in a buffer containing 12.5 mM Hepes (pH 7.4), 0.25 mM EDTA, and 35 μM DTT in the presence of 10 mM DTT, 80 μM H₂O₂, 320 μM H₂O₂ or 1 mM pervanadate for 30 min at room temperature (R.T.). To measure reversibility of oxidation, aliquots from these reactions were incubated further in the presence of 10 mM DTT and 5U of catalase at R.T. for 3 h. Catalytic activity was then measured by using pNPP as substrate and pre-incubated PTP1B (125 ng) in 200 μl of phosphatase assay buffer (25 mM Hepes pH 7.4, 50 mM NaCl, 10 mM pNPP) for 5 min at 37°C. For in vitro dimedone-labeling, purified PTP1B (0.25 μg μl⁻¹) was incubated for 5 min at R.T. in the presence or absence of 5 mM dimedone in labeling buffer (12.5 mM Hepes, pH 7.4, 0.25 mM EDTA and 35 μM DTT) containing a final concentration of 10 mM DTT, 80 μM H₂O₂, 320 μM H₂O₂ or 1 mM pervanadate, as indicated. Two microliters of each reaction were diluted into 148 μl of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and subjected to SDS-PAGE, followed by immunoblotting with anti-dimedone-Cys and anti-PTP1B antibodies. For post-oxidation dimedone-labeling, purified PTP1B (0.5 μg μl⁻¹) was first incubated in labeling buffer with 10 mM DTT, 80 μM H₂O₂, or 320 μM H₂O₂ at R.T. for 30 min, and then further incubated for 5 min at R.T. after addition of an equal amount of 10 mM dimedone solution containing the same concentration of DTT or H₂O₂.