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.