β3 tubulin

The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.: 1:1000; endocytosis assay: 1:25), antibodies against GluN2A (alomone labs; 1:500) and GluN1 (Synaptic Systems; 1:200), anti pGluN2BTyr1472 antibody (AAT Bioquest; WB 1:500); mouse (ms) antibodies against GluN2B (NeuroMab; WB 1:500), PSD-95 (NeuroMab; ICC 1:1000), Map2 (Sigma-Aldrich; ICC 1:2000), β3-tubulin (Synaptic System; WB 1:1000); rat antibody against β1-integrin CD29 (BD Pharmingen; 1:25).
Fluorescently labelled secondary antibodies that were used for ICC against rabbit, mouse, guinea pig were purchased from Invitrogen conjugated with either Alexa 488, 568, 647 (1:1000) or from Dianova conjugated with Cy3, Cy5 (1:1000). Fluorescently labelled secondary antibodies against ms, rb and guinea pig for quantitative immunoblotting were purchased from Invitrogen (ms Alexa Fluor 680, 1:20,000) and from Rockland (rb IRDye 800 W, 1:20,000).
Hyaluronidase (Hya, Sigma-Aldrich) was used at 100 units/ml, and TTX (0, 5 µM), CNQX (5 µM), Biccuculine (BCC) (10 µM), AP5 (10 µM), Ifenprodil (3 µM) was purchased from Tocris. All other chemicals and drugs were purchased from Sigma-Aldrich (USA).

For immunolabellings shown in Figs. 3 and 4, U-2 OS cells were seeded onto fibronectin-coated coverslips and processed essentially as described^{89 (link)}. Briefly, cells were fixed with pre-warmed 4% PFA (paraformaldehyde) containing 0.1% glutaraldehyde in PBS for 20 min and extracted with 0.1% Triton X-100 in PBS for 1 min. Primary antibodies were α-tubulin (clone DM1A, T9026, Sigma-Aldrich, 1:300 dilution, AB_477579) and acetylated α-tubulin (6-11B-1, T6793, Sigma-Aldrich, 1:2000 dilution, AB_477585). Neuronal progenitor cells shown in Fig. 1 were plated onto pre-coated coverslips as described above, and treated for 6 h with neuron medium containing DMSO as control, 25 µM kainic acid (Sigma-Aldrich, K0250), 50 µM SMER28, 150 nM paclitaxel or combinations of these, as indicated. SMER28 was added 16 h prior to the treatment with kainic acid. Neuronal progenitors were fixed with 4% PFA containing 0.25% glutaraldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 1 min and stained with β-3-Tubulin (#302302, Synaptic Systems, 1:300 dilution, AB_10637424) antibodies. Cells were mounted in ProLong Diamond Antifade (Invitrogen).