Immunoprecipitation (IP) of endogenous CRMP2
CAD cells were incubated overnight with vehicle (0.1 % DMSO) or CBD3063 (20 µM). The next day the cells were lysed into the IP buffer containing 20 mM Tris-HCl pH=7.4, 50 mM NaCl, 2 mM MgCl2, 10 mM N-Ethylmaleimide (NEM), 1% (vol/vol) NP-40, 0.5% (mass/vol) sodium deoxycholate, 0.1% (mass/vol) sodium dodecyl sulfate (SDS) with protease inhibitors (Cat# B14002, Biotool, Houston, TX), phosphatase inhibitors (Cat# B15002, Biotool, Houston, TX) and Nuclease (Cat# 88701, Thermo Fisher Scientific, Waltham, MA). Total protein concentration was determined by BCA protein assay kit (Cat# PI23225, Thermo Fisher Scientific, Waltham, MA). Five hundred micrograms of total protein were incubated overnight with 2 μg of CRMP2 antibody (Cat# C2993, Sigma-Aldrich, St. Louis, MO) at 4°C under gentle agitation. Protein G magnetic beads (Cat# 10004D, Thermo Fisher Scientific, Waltham, MA), pre-equilibrated with the immunoprecipitation buffer, were then added to the lysates and incubated for 2 h at 4°C to capture immuno-complexes. Beads were washed four times with IP buffer to remove nonspecific binding of proteins, before resuspension in Laemmli buffer and boiling at 95°C for 5 min prior to immunoblotting.