Mouse anti gephyrin

Antibodies used are as follows: rabbit anti-GFP (1:500; Thermo Fisher Scientific, Invitrogen, A11122); rabbit anti-GFP polyclonal antibody, Alexa Fluor 488 (1:500; Thermo Fisher Scientific, no. A21311); rabbit anti-Cux1 (1:500; Santa Cruz Biotechnology, sc-13024 X); mouse anti-human Rorβ (1:300; Perseus Proteomics, PP-N7927-00); rabbit anti-Ctip2 (1:250; Abcam, ab240636); guinea-pig anti-Vglut1 (1:500; Merck Millipore, AB5905); mouse anti-NeuN (1:500; Chemicon, MAB377; clone A60); guinea pig anti-Vglut2 (1:1000; Sigma-Aldrich, Merck Millipore, AB2251-I); mouse anti-gephyrin (1:250; Synaptic Systems, no. 317005); mouse anti–synaptotagmin-2 (1:250; ZFIN, no. ZDB-ATB-081002-25); mouse monoclonal anti-PV (1:500; Sigma-Aldrich, Merck Millipore, no. p3088); goat anti-mouse Alexa Fluor 405 (1:200; Thermo Fisher Scientific, no. A-31553); goat anti-mouse Alexa Fluor 488 (1:500; Life Technologies, no. A-11029); goat anti-rabbit Alexa Fluor 488 (1:500; Life Technologies, no. A-11034); goat anti-rabbit Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A-A21245); goat anti-mouse Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A21236); and goat anti–guinea pig Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A21450).

Differential centrifugation was performed as described^{77 (link),78 (link)}. In brief, 16 million cortical neurons were lysed in homogenizing buffer (HB; 150 mM KCl, 50 mM Hepes pH 7.4, 1x complete protease inhibitor [Roche], 5 µL Ribolock [ThermoFisher] per 10 mL HB) on ice. Homogenate was spun at 16,000 × g for 10 min at 4 °C (S16, P16). Supernatant S16 was then spun at 100,000 × g for 20 min at 4 °C (S100, P100). When required, samples were treated with RNase1 (Ambion) prior to centrifugation. P100 pellets were volume-even resuspended in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 0.5% (w/v) sodium deoxycholate, 1 vol% NP-40, 0.1% (w/v) SDS, 1× complete protease inhibitor, Roche) at 37 °C. All fractions (S16, P16, S100, P100) were methanol/chloroform extracted as described^{76 (link)}. Samples were analyzed by Western blot, using polyclonal goat anti-DDX6 1:1,000 (Abnova) and mouse anti-Gephyrin 1:1,000 (Synaptic Systems) primary and donkey anti-goat IRDye 680RD conjugated 1:10,000 and donkey anti-mouse IRDye 800CW conjugated 1:10,000 (LI-COR) secondary antibodies.

Western blot analysis was performed as described^{75 (link)}. In brief, protein samples were transferred on a nitrocellulose membrane and blocked in 2%(w/v) BSA. Proteins were detected using rabbit anti-Rck 1:1000 (MBL), goat anti-DDX6 1:1,000 (Abnova), mouse anti-ß-actin 1:5000 (Sigma-Aldrich), rabbit anti-RPL7A 1:1000 (Abcam), mouse anti-Gephyrin 1:1000 (Synaptic Systems) and mouse anti-GFP 1:500 (self-made, kind gift by Angelika Noegel, Köln) primary antibodies and donkey anti-rabbit IRDye 680RD conjugated 1:10,000, donkey anti-mouse IRDye 800CW conjugated 1:10,000 and donkey anti-goat IRDye 680RD conjugated 1:10,000 (LI-COR) secondary antibodies. Primary antibody binding was detected using the LI-COR Odyssey IR scanner.

Syt4, Mef2c,
Kif1a and Rac3 cDNA was cloned
into pcDNA3.1(−) (Thermofisher Scientific).
Tbr1^layer5 mutant cells were
transfected with Syt4, Mef2c,
Kif1a, Rac3 expression vectors and
Tbr1^wild-type were transfected with
mock empty vector using Lipofectamine 3000 (Invitrogen) for 6 hr.
Following incubation, the media was replaced by Neurobasal medium
containing B27 supplement, Penicillin/Streptomycin, 25% glucose, and
glutamax. Cultures were grown for 14 days in vitro.
After 14 days, cultures were washed 3 times with 0.5 mL 1X PBS for 5 min
each and fixed for 15 min with 4% PFA in 1X PBS at RT. Fixed cells were
washed 3 times with 0.5 mL 1X PBS and blocked in 1X PBS containing 10%
Normal Serum, 0.1% Triton X- 100 and 2% BSA for 1 hr at RT. Primary
antibodies including mouse anti-Vglut1 (1:200, Synaptic Systems) and
rabbit anti-PSD95 (1:200, Cell Signaling; excitatory synapses), rabbit
anti-Vgat (1:500, Synaptic Systems) and mouse anti-gephyrin (1:200,
Synaptic Systems; inhibitory synapses) were diluted 1:200 in blocking
solution. Cells were stained for excitatory and inhibitory synapses with
primary antibodies for 48 hr at 4°C with gentle shaking. On a
shaker, the cells were washed 3 times with 0.5 mL 1X PBS for 5 min each
and incubated with the secondary antibody for 2 hr (room temperature),
washed 3X with 1X PBS, and mounted. This experiment was repeated twice
(n = 2).