Map1b

Antibodies against ATF2, MAP1B, Tau, Ankirin G, β-catenin, GSK-3 β(ser9), β-actin, MAP2 and phalloidin were obtained from Cell Signaling Technology, Inc. (Trask Lane, Danvers, MA). Neurobasal growth medium was obtained from Life Technologies (Carlsbad, CA). Other reagents were purchased from Sigma (St. Louis, MO). Wnt-C59 inhibitor was dissolved in DMSO and added to neuronal cultures at a final concentration of 0.01% DMSO or less (Cellagen Technology, San Diego, CA). Recombinant Wnt3a, Wnt5a, and Wnt7a (R&D Systems, Abingdon, UK) were used at 50 ng/ml, 100 ng/ml, and 200 ng/ml, respectively with corresponding dilutions in vehicle.

At the indicated time, neurons on coverslips were rinsed briefly with D-PBS and fixed by a sequential paraformaldehyde/methanol fixation procedure^{69 (link)}. The following antibodies were used for immunostaining: primary antibodies to tubulin α-subunit (mouse monoclonal 12G10, 1:1000 dilution; Developmental Studies Hybridoma Bank, University of Iowa, IO), ankyrin G (rabbit polyclonal H-215, 1:50 dilution; Santa Cruz Biotechnology Inc., CA), MAP2 (mouse monoclonal clone HM-2; 1:500; Sigma, MO), Tau (rabbit polyclonal LF-PA0172; 1:500; Abfrontier, Seoul, Korea), Hnrnpa2b1 (Goat polyclonal, 1:500; Santa Cruz Biotechnology Inc., CA), Map1b (rabbit polyclonal; 1:500; Cell Signaling Technology, Danvers, MA), and secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG [1:1,000], Alexa Fluor 568-conjugated goat anti-rabbit IgG [1:1,000], Alexa Fluor 568-conjugated rabbit anti-goat IgG [1:1,000], Molecular Probes, OR). Neurons were then incubated with primary antibodies followed by secondary antibodies and mounted on slides as previously described⁷⁴ .