Bassoon

Antibodies: Mover (Synaptic Systems, 248003); Tubulin (Sigma, DM1A); myc-tag (Santa Cruz, clone 9E10); Synapsin-1 (Synaptic Systems, clone 46.1); Synaptophysin (Sigma, SVP-38); GFP (Abcam, cat. GFP6556, for immunofluorescence); GFP (Synaptic Systems, 132002, for Western blots); Bassoon (ENZO Life Sciences, sap7f407). Secondary antibodies (Invitrogen): anti-mouse Alexa 647 1:1,000; anti-mouse Cy3 1:500; anti-guinea pig Alexa 647 1:1,000; anti-rabbit Alexa 647 1:1,000. All mammalian expression constructs carried the CMV promotor. All recombinant Mover sequences were from rat Mover. For tagging with GFP, the A206K mutant of EGFP was used, which prevents dimerization of EGFP.

Neurons at DIV 14–15 were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 and, blocked with 2% normal goat serum and 0.1% Triton X-100 in PBS. The primary antibodies used were MAP2 (1:20000, Abcam, Cat. No. ab5392), SMI-312 (1:5000, Abcam, Cat. No. ab24574), VAMP2/synaptobrevin (1:1000, Synaptic Systems, Cat. No. 104 211), Bassoon (1:500, Enzo Life Science, Cat. No. SAP7F407), Synaptophysin-1 (1:1000, SynapticSystems, Cat. No. 1011004), VPS35 (1:500, Abcam, Cat. No. ab97545), GluA1 (1:50, Merk Millipore, Cat. No. MAB2263). The secondary antibodies were conjugated to Alexa dyes (1:1000, Molecular Probes). The cells were mounted on microscope slides with Dabco-Mowiol (Invitrogen). Image acquisition was performed on a Carl Zeiss LSM510 confocal microscope, with a Plan-Neofluar 40×/1.3 oil objective. Colocalization analysis was performed using JACoB plugin in zoomed neurites^{54 (link)}. Morphological analysis was performed using SynD^{36 (link)}.

Primary antibodies used for immunocytochemistry were all diluted 1:200, for western blotting a dilution of 1:500 was used (except for Actin which was diluted 1:100000). The Shank2 (“ppI-SAM pabSA5192”) and Shank3 antibodies (“PRC pab,” simply referred to as “Shank3” in the study and “C-term/ProSAP2/Shank3”) have been characterized previously (Bockers et al., 1999 (link); Bockmann et al., 2002 (link); Schmeisser et al., 2012 (link)). The following primary antibodies were purchased from commercial suppliers: Actin (Sigma-Aldrich Cat# A2228 RRID:AB_476697), Bassoon (Enzo Life Sciences Cat# ADI-VAM-PS003-F RRID:AB_11181058), CTIP2 (Abcam Cat# ab18465 RRID:AB_2064130) GAD65 (Abcam Cat# ab85866 RRID:AB_1860505), Homer 1b/c (Synaptic Systems GmbH Cat# 160 023, no RRID yet), GluN1 (Sigma-Aldrich Cat# G8913 RRID:AB_259978), Shank1 (Novus Cat# NB300-167 RRID:AB_2187584), SPO (Synaptic Systems GmbH Cat# 102 002 RRID:AB_887841), Syn1/2 (Synaptic Systems GmbH Cat# 160 003, no RRID yet), VGLUT1 (Synaptic Systems GmbH Cat# 135 304 RRID:AB_887878), VGLUT2 (Synaptic Systems GmbH Cat# 135 404 RRID:AB_887884), and VGLUT 3 (Synaptic Systems Cat# 135204, no RRID yet).