Figure 3

AsCy5 targets and activates actHePTP in complex proteomes and cells. (A) Activation of actHePTP in cell lysates. Clarified lysates (0.125 mg/mL) from E. coli expressing wt-HePTP or actHePTP were incubated with the indicated concentrations of AsCy5 for 60 minutes, then assayed for PTP activity with pNPP (1.5 mM, quenched assay). (B) Visualization of targeted proteins from AsCy5-treated cell lysates. Clarified lysates (0.085 mg/mL) expressing actHePTP or wt-HePTP were incubated with the indicated concentrations of AsCy5 for 15 minutes. The resulting solutions were diluted by a factor of 25, and 15 µL of each dilution were separated by SDS-PAGE. AsCy5-bound proteins were visualized using near infrared illumination. An uncropped image of the gel is presented in Supplementary Fig. S8. (C) Activation of actHePTP in intact cells. E. coli cells expressing either wild-type HePTP (wt) or actHePTP were incubated with the indicated concentrations of AsCy5 for 2 hours, washed thoroughly, and lysed. PTP activities of the resulting lysates were measured with pNPP (2 mM, quenched assay).