Immunoblotting

Frozen tissues were lysed in L-RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, pH 7.2 containing 1 mM Na₃V0₄, 1 mM PMSF, 10 μg/mL aprotinin, 1 μg/mL leupeptin). Equal amounts of protein were immunoblotted with antibody to SH2B1 (αSH2B1) (sc-136065, RRID:AB_2301871; Santa Cruz Biotechnology) (1:1,000 dilution) or β-tubulin (sc-55529, RRID:AB_2210962; Santa Cruz Biotechnology) (1:1,000 dilution) as described in Joe et al. (19). For immunoprecipitations, tissue lysates containing equal amounts of protein were incubated with αSH2B1 (1:100) and immunoprecipitated and immunoblotted as in Joe et al. PC12 cells (ATCC) were cultured and treated as in Joe et al. Briefly, the cells were grown in PC12 medium A (RPMI medium, 5% FBS, 10% heparan sulfate) in 10-cm dishes coated with rat tail type I collagen (#354236; Corning). Cells were transfected and, 24 h later, incubated overnight in deprivation medium (RPMI medium, 2% heparan sulfate, 1% FBS) before being lysed and immunoblotted with αSH2B1.