Anti β3 tubulin

Immunofluorescent stainings were performed according to standard protocols [13 (link)]. In brief, samples were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X100 and blocked in a mix of 1% BSA and 5% goat serum. Primary antibodies were then employed as follows: anti-microtubule associated protein 2 (MAP2) (1:2000, Cat No. ab5392, Abcam) and anti-β3-tubulin (alias TUJ1) (1:500, Cat No. T5076, Sigma Aldrich, St. Louis, MO, USA) served as general neuronal markers and anti-neurofilament heavy chain (1:300, Cat No. smi-32P, BioLegend, San Diego, CA, USA) to detect heavy neurofilament H as MN marker (SMI32), anti-acetylated tubulin (1:400, Cat No. T7451, Sigma Aldrich, St. Louis, MO, USA) and anti-α tubulin (1:1000, Cat No. ab4074, Abcam, Cambridge, UK) to elucidate post-translational modification of β3-tubulin and α-tubulin, respectively, and anti-cleaved-caspase-3 (1:500, Cat No. ab32042, Abcam, Cambridge, UK) to determine apoptosis. The nuclei were counterstained with Hoechst 33342 (Cat No. 14533, Sigma Aldrich, St. Louis, MO, USA).

A rabbit antiserum directed against the Shank1 PDZ domain (recognizing all Shank variants) has been described previously [15] (link). For generation of antisera against protein fragments derived from the 5′ region of the Shank1 mRNA, corresponding cDNA sequences were cloned into pGEX vectors in frame with the glutathione-S-transferase (GST) coding sequence. GST fusion protein was expressed using standard methods, and used for custom immunization of rabbits (Biogenes GmbH, Berlin, Germany). Anti-monomeric red fluorescent protein (mRFP) has been described before [24] (link). Anti-neomycin phosphotransferase was obtained from Upstate/Biomol (Hamburg, Germany); anti GluR1, anti-eIF2a and phospho-eIF2a from Abcam Cambridge, UK; anti-β3 tubulin, anti-Shank1 and anti-MAP2 were from Sigma.