Overnight culture of Escherichia coli BL21 (DE3; pGEX-PTP1B and pGEX-PTP1B-D181A) was diluted 1:10 with LB containing 100 μg/mL ampicillin grown for 1 hour at 37°C and induced for another hour by the addition of isopropyl β-D-1-thiogalactopyronoside to 1 mM. Bacteria were sonicated three times for 20 seconds each time in lysis buffer containing 10 mM imidazole-HCl (pH 7.2), 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, and 1% Triton X-100. Lysates were clarified by centrifugation, and the supernatants were incubated with 500 μL of 50% glutathione-coupled beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 30 minutes at 4°C. GST-PTP1B fusion proteins were washed in lysis buffer and eluted twice with 1 mL of 5 mM reduced glutathione (Sigma) in phosphatase buffer (20 mM Tris [pH 7.4], 5% glycerol, 0.05% Trion X-100, 2.5 mM MgCl2, aprotinin [2 μg/mL], and leupeptin [5 μg/mL]). Glycerol was added to a final concentration of 33% (vol/vol), and aliquots of enzyme were stored at −20°C.