Immunoblot preparation and analysis
Indicated samples were loaded on 4–20% Novex gels (cat. no. XP04205BOX; Thermo Fisher Scientific, Waltham, MA). Proteins were transferred to preactivated PVDF membranes for 1 h at 100 V using TGS [25 mM Tris, pH 8.5, 192 mM glycine, 0.1% (mass/vol) SDS], 20% (vol/vol) methanol as transfer buffer (0.45 μm; Cat# IPVH00010; Millipore Sigma, St. Louis, MO). After transfer, the membranes were blocked at room temperature for 1 h with TBST (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) with 5% (mass/vol) nonfat dry milk, and then incubated overnight at 4 °C separately with indicated primary antibodies, βIII-Tubulin (Cat# G7121; Promega, Madison, WI), CRMP2 (Cat# C2993; Sigma-Aldrich, St. Louis, MO), Cav2.2 (Cat# TA308673; Origene, Rockville, MD), CRMP2 pSer522 (Cat# CP2191; ECM Biosciences, Versailles, KY), CRMP2 pThr514 (Cat# PA5-110113; Invitrogen, Waltham, MA), in TBST, 5% (mass/vol) BSA. For examining the effect of CBD3063 on CRMP2 phosphorylation state, CAD cells were treated overnight with vehicle (0.1 % DMSO) or CBD3063 (20 µM) and the next day cells were lysed using RIPA buffer. Approximately 40 μg of total proteins were loaded on an SDS-PAGE and then transferred to polyvinylidene difluoride membranes and blocked at room temperature for 1 hour. Primary antibodies used for probing were CRMP2 (Cat# C2993, Sigma-Aldrich, St Louis, MO), CRMP2 pThr514 (Cat# PB-043, Kinasource, Dundee, Scotland, United Kingdom), CRMP2 pSer522 (Cat# CP2191, ECM Biosciences, Versailles, KY), and CRMP2 pT555 (Cat# CP2251, ECM Biosciences, Versailles, KY). Following incubation in HRP-conjugated secondary antibodies from Jackson Immuno Research (West Grove, PA), blots were revealed by enhanced luminescence (WBKLS0500; Millipore Sigma St. Louis, MO).