Optimizing STEP Substrate Tyr Phosphorylation

Wild-type, male C57BL/6 mice (3–6 mo) were used for all studies. An initial dose–response curve was carried out using S₈ (0.5, 1, and 3 mg/kg, i.p.) or TC-2153 (1, 3, 6, and 10 mg/kg, i.p.). Pilot studies were conducted to optimize the time after i.p. injection when STEP substrates showed maximum Tyr phosphorylation (1–3 h). Cortical tissues were dissected out 3 h postinjection and processed for subcellular fractionation. We homogenized brain tissue in buffer containing (in mM): 10 Tris-HCl, pH 7.6, 320 sucrose, 150 NaCl, 5 EDTA, 5 EGTA, 20 NaF, 1 Na₃VO₄, and protease inhibitors (TEVP). Homogenates were centrifuged at 800 × g to remove nuclei and large debris (P1). Synaptosomal fractions (P2) were prepared from S1 by centrifugation at 9,200 × g for 15 min. The P2 pellet was washed twice and was resuspended in TEVP buffer. In some experiments, mice were injected with S₈ (1 mg/kg, i.p.) or TC-2153 (3 mg/kg, i.p.), and cortex, cerebellum, and spleen were removed to test for the in vivo inhibition of the highly related PTPs, HePTP, and PTP-SL [57] (link)–[60] (link).