Smi312 smi 312r

Fluorescence ICC was performed according to procedures previously described^{47 (link)}. The following primary antibodies were used to assess neuronal differentiation: TuJ1 (T8660, Sigma), MAP2 (CPCA-MAP2, EnCor Biotechnology, Gainesville, FL, USA), SMI312 (SMI-312R, Covance, Princeton, NJ, USA), and GFAP (MCA-5C10; EnCor Biotechnology). To evaluate CRMP2 expression and phosphorylation levels with ICC, the same p-CRMP2^T514 and CRMP2 antibodies from Cell Signaling Technologies as for western blot analysis were used. For the high-throughput high-content screening assay, neuronal cultures were co-immunostained for p-CRMP2^T514, the dendritic marker MAP2, and the nuclear protein HDAC2 (05-814, EMD Millipore, Burlington, MA, USA).

TPX2 antibody was a kind gift from Oliver Gruss24 (link). Anti-RanGTP antibody (AR-12) was a kind gift from Ian Macara37 (link). Mouse anti-α-tubulin monoclonal antibody DM1A (T6199) and Duolink reagents were from Sigma (St Louis, MO). Mouse anti-β-III-tubulin antibody TUJ1 (MMS-435P) and mouse anti-neurofilament monoclonal antibody SMI312 (SMI-312R) were from Covance (Princeton, NJ). Mouse anti-γ-tubulin antibody GTU-88 (GTX11316), HRP-labeled goat-anti-rabbit IgG antibody (GTX213110), and HRP-labeled goat-anti-mouse IgG antibody (GTX213111) were from GeneTex (Irvine, CA). Mouse anti-actin antibody C4 (MAB1501), mouse anti-GAPDH antibody 6C5 (MAB374), and rabbit anti-MAP2 polyclonal antibody (AB5622) were from Millipore (Billerica, MA). Rabbit anti-Ran polyclonal antibody (ab31118) and rabbit anti-importin β1 (ab45938) were from Abcam (Cambridge, UK). Mouse anti-importin-α antibody (sc-55538) was from Santa Cruz Biotechnology (Santa Cruz, CA). Dylight-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Alexa Fluor-conjugated secondary antibodies were from Life Technologies (Carlsbad, CA).