Syntaxin

hES-MN cultures (day 35) were fixed in 4 % paraformaldehyde-PBS for 10 min and permeabilized with 0.1 % Triton X-100. After being blocked with 10 % serum, the cells were then incubated overnight with the following primary antibodies: Tau5 (Thermo Scientific), LIM3 (Millipore), ChAT (Millipore), NeUN (Millipore), Hb9 [Developmental Studies Hybridoma Bank (DSHB)], Isl1 (DSHB), and β-III tubulin (Covance) (as shown in Fig. 1b), as well as antibodies for SNAP-25 (BD Biosciences), SV2A (Millipore), Syntaxin (Sigma), VAMP-2 (R&D Systems), and MAP2 (Millipore) (as shown in Fig. 2b) in PBS with 10 % serum according to the manufacturers’ suggested working concentrations. On the following day, appropriate secondary antibodies, conjugated with Alexa488 and Alexa594, were incubated with the cells for 2 h at room temperature. Image acquisition was performed using Zeiss or Opera (PerkinElmer) confocal microscopes.

Protein levels were measured by the Bradford method. For the cross-linking experiment, 20 μg of protein from each rat was loaded in each lane of a 4–15% Tris-HCl polyacrylamide gradient gel (Bio-Rad) for electrophoresis. For analysis of the PSD fractions, 5 μg of protein was loaded in each lane of a 10% Tris-HCl polyacrylamide gel. Following transfer, membranes were incubated in blocking solution [5% non-fat milk in Tris buffered saline containing Tween 20 (TBS-T buffer)] for 1 hour at room temperature. Membranes were then incubated overnight at 4°C in primary antibody [GluA1, 1:500; GluA2, 1:5000; pGluA1(S831), 1:500; pGluA1(S845), 1:1000; TH, 1:2000; Millipore; β-actin, 1:10000; CaMKIIα, 1:5000; Syntaxin, 1:5000; Sigma-Aldrich; pCaMKIIα(T286), 1:1000; NR2b, 1:1000; PSD95, 1:2000; Stargazin, 1:1000; Cell Signaling; CaMKII β, 1:1000; Invitrogen; PKA, 1:500; AKAP150, 1:5000; Santa Cruz]. After extensive washing in TBS-T buffer, membranes were then incubated in an HRP-conjugated secondary antibody (anti-rabbit, 1:10000; anti-mouse, 1:10000; Upstate) for 60 minutes followed by further extensive washing. The protein bands were visualized using a chemiluminescence detection system (ECL Advanced). Blots were digitally imaged using the GeneSnap Bio Imaging System and quantified using Genetool software (Syngene).

Eyes were enucleated and fixed by immersion in freshly prepared 4% paraformaldehyde in PBS for 3 h on ice. For adult eyes, a small incision was made along the ora serrata after 10 min in paraformaldehyde, to allow better access to the fixative. The eyes were then rinsed in PBS and cryo-protected in sucrose 20% overnight, frozen and embedded in a sucrose:optimal cutting temperature (O.C.T.) compound (1:1) mix. For immunofluorescence, retinas were sectioned and processed for staining on the same day. Slides were preincubated for 1 h in blocking buffer (20% goat serum in 0.2% Triton) and then incubated 48 h at 4 °C with primary antibodies diluted in PBS. Primary antibodies used were PNPLA6 (NTE; 1/100, Abcam), Na²⁺K⁺-ATPase (1/100, Thermo Scientific), Syntaxin (1/500, Sigma), NF-165 (1/200, gift from B. Barres, Stanford U.) and Brn3b (1/100, Santa-Cruz). Bound antibodies were detected using appropriate secondary antibodies conjugated with Alexa 488, 555, 594 or 647 (1:1,000; Invitrogen) diluted in antibody buffer at room temperature for 1 h. In all cases, nuclei were counterstained with Hoechst 33342 (Molecular Probes).