β 3 tubulin tubb3

CxV explants were fixed at 4–6 div for 10 min in 4% PFA (Sigma), washed with PBS and permeabilized for 10 min with 0.2% Triton-X in PBS. The following primary antibodies were used: Gad67 (Chemicon), GAP-43 (Abcam), GFAP (Sigma), mO4 (gift from J. Trotter), PKCγ (Santa Cruz Biotechnology), phospho-GAP-43 (Thermo Fischer), phospho-PKCγ (Biozol), PTEN Cascade Biosciences), PV (Swant), SMI-32 (Covance), and β-III-Tubulin (Tubb3, mouse, Covance; rabbit, Abcam). For human neuronal cultures IL-4R (BD Pharmingen), Homer-1/2/3 (Synaptic Systems), IRS1 (Abbexa), phospho-IRS1 (pIRS1, Abbexa), NeuN (Merck Millipore), PKCγ (Abcam), Neurofilament light chain (Abcam), MAP2 (Abcam), Synaptophysin (Synaptic Systems), and Tubb3 (BioLegend), were used in addition. After washing with PBS, incubation was performed for 1 h with Alexa-conjugated anti-mouse and anti-rabbit secondary antibodies (Life Technologies), Alexa 488-conjugated Phalloidin (Thermo Fisher) and dapi (Invitrogen).

Reagents were from the following companies: Hoechst (B2261), MTT (M2128), PF-573228 (PZ0117), polybrene (H9268) puromycin (P7225), and X-Gal (B4252) from Merck Sigma-Aldrich (Darmstadt, Germany); and Defactinib (VS-6063, PF-04554878) from Selleckchem (Houston, TX, USA).
Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).