Homer1

iPSC derived neurons were grown in a black 96 well half area plates with transparent bottom (Greiner) and fixed in the plate with 4% paraformaldehyde (PFA) for 5 min. Cells were permeabilised with 0.05% Saponin for 20 min and blocked for 30 min in PBS containing 10% goat serum at RT, before incubation with primary antibodies overnight at 4°C in PBS containing 0.1% Tween and 1% goat serum. Antibodies used as follows: Map2 (Abcam, #AB92424, 1:1000), Homer1 (Synaptic Systems, #160003, 1:500) and Synapsin I/II (Synaptic Systems, #106004, 1:500). AlexaFluor-conjugated secondary antibodies were incubated for 1 h at RT in PBS containing 0.1% Tween. Nuclear DNA was stained with DAPI (Thermo Fisher Scientific) for 5 min at RT. Images were captured by the Opera Phenix Plus High Content Screening System (Perkin-Elmer).

Following drug treatment, cells were fixed either in methanol or in 4% paraformaldehyde (PFA) and immunostaining was performed with primary antibodies specific for the DNMT3AN-terminus, CUL4B (Proteintech, catalog #12916-1-AP), GFAP (Synaptic Systems, catalog #173011), HOMER1 (Synaptic Systems, catalog #160011), NEDD8 (Proteintech, catalog #16777-1-AP), MAP2 (Sigma-Aldrich, catalog #M4403), 5meCytosine (Calbiochem, catalog #MABE146, clone 33D3), Synaptophysin1 (Synaptic Systems, catalog #101004); secondary antibodies anti-rabbit/mouse-Alexa Fluor 488/568 linked (Molecular Probes Europe BV, Leiden, The Netherlands), anti-rabbit/mouse-Cy^TM5-conjugated (Dianova, Hamburg, Germany) and DAPI (Sigma-Aldrich, catalog #D9564). Live immunostaining was performed for the detection of the surface expression of the GluN2A subunit of NMDARs. To this end, the GluN2A antibody (Alomone Labs, #AGC-002) was applied in cell media for 20 min, then cells were fixed and staining was performed as outlined above.

Rab10 (4262S, Cell Signaling Technology) for immunoblot, Rab10 (ab237703, Abcam) for immunofluorescence, pRab10 for immunoblot (ab230261, Abcam), pRab10 for immunofluorescence (ab241060, Abcam), Hsc70 (ab19136, Abcam), α-Tubulin (SC23948, Santa Cruz Biotechnology), parvalbumin, (NBP2-50036, NovusBio), NeuN (MAB377, Millipore), SATB2 (ab51502 Abcam), calretinin (MAB1568, Millipore), DARPP32 (MAB4230, R&D Systems), ChAT (NBP2-46620, NovusBio), DAT loop (6-8D6 Santa Cruz Biotechnology), tyrosine hydroxylase (TH) (ab76442, Abcam), CD68 (NBP2-33337SS, NovusBio), GFAP (AB5541, Millipore), Olig2 (MABN50 Millipore), KDEL receptor (sc-58774, Santa Cruz Biotechnology), TGN46 (MA3-063, ThermoFisher), LAMP1 (1D4B, DHSB), EEA1 (NBP2-36568, NovusBio), α-Synuclein (610786, BD Transduction Lab), Synuclein (ab51252, Abcam), VAMP2 (104 211, Synaptic Systems), Homer1 (160 006 Synaptic Systems), Rab8a (ab188574).

Primary hippocampal cultures were prepared as described above. After 18–19 DIV, the cells were incubated with 20 µM BIC, 1 µM TTX or no drug (Ctrl) for 1, 3 or 7 days as indicated, subsequently washed with ice-cold PBS (Invitrogen) and scraped in PBS supplemented with 0.5% SDS, 0.5% Triton-X100, protease inhibitor cocktail (Roche), Halt phosphatase inhibitor (Thermo Scientific) and benzonase (2 µL/mL, ≥250 units /µL, Sigma). The lysates were cleared by centrifugation at 17,000 x g for 15 min. Protein concentration was determined by BCA assay (Thermo Scientific). Equal protein amounts were used for western blot analysis. Proteins were separated by electrophoresis and immunoblotted with antibodies against Bdnf (1:400, Santa Cruz Biotechnology, Ref: sc-546), Homer1 (1:1000, Synaptic Systems, Ref: 160003), GluA1 (1:1000, Synaptic Systems, Ref: 182003), Synaptopodin (1:1000, Synaptic Systems, Ref:163002), Camk1g (1:2000, Abcam, Ref: ab227209), β-actin (1:3000, Sigma, Ref: A5316), eIF2α (1:5000, Cell Signaling Technology, Ref: L57A5) and p-eIF2α (1:2500, Invitrogen Ref: 44728G). For western blots against eIF2α and p-eIF2α, phosphatase inhibitor was added to all solutions. Three biological replicates were performed.

Primary antibodies used for immunocytochemistry and/or western blots included: Homer1 (1:750, Synaptic Systems; 160003), Shank2 (1:1,000, Synaptic Systems; 162204), Shank3 (1:500, Synaptic Systems; 162302 and 162304), GluA1 and GluA2 [1:8 for surface staining and 1:100 for whole cell staining, Millipore; PC246 and MAB397], VGluT1 (1:100, NeuroMab; N28/9), Shank2 (1:100, Neuromab; N23B/6), Shank3 (1:100, NeuroMab; N367/62), PSD-95 (1:100, NeuroMab; K28/43), MAP2 (1:5,000, Abcam; ab5392), green fluorescent protein (1:1,000, Abcam; ab13970), actin (1:1,000, Abcam; ab8227), and Shank2 (1:250, Cell Signaling; 12218). A custom-made VGluT1 antibody (1:500, polyclonal rabbit) was generously provided by Dr. Richard Reimer (Stanford University). All secondary antibodies (1:500, A11029, A11034, A11036, A11039, A11041, A11075, A21235 and A21449) were obtained from Life Technologies with the exception of the Dylight-350 antibody (1:250, Thermo Fisher; SA5-10069) and the HRP-conjugated antibodies (1:10,000, rabbit, mouse or guinea pig; Jackson ImmunoResearch; 706-035-148, 115-035-003 and 111-035-144).

DIV13 neurons in 24-well plates were fixed with warm 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS for 7 min. Next, PFA was removed and cells were washed 3× with PBS then blocked in a buffer containing 0.2% Triton X-100 (Roche) in 50% normal goat serum (NGS; Jackson ImmunoResearch)/50% antibody buffer (PBS containing 1% BSA, 0.04% NaN3, 0.2% Triton X-100) at room temperature (RT) for 30 min. Following another 3× PBS wash, cells were treated with 10% NGS/90% antibody buffer containing primary antibodies against Bassoon (1:500; mouse; Enzo/Assay Designs) and Homer1 (1:500; rabbit; Synaptic Systems), at 4°C overnight in the dark. The next morning, another 3× PBS wash was performed, followed by a 2-h RT incubation in 10% NGS/90% antibody buffer containing the following fluorescently-conjugated secondary antibodies: goat anti-mouse Alexa Fluor 488 (1:500; Invitrogen) and goat anti-rabbit Alexa Fluor 594 (1:500; Invitrogen). After final round of 3× PBS washes, coverslips were transferred to glass slides with Vectashield mounting medium containing DAPI (Vector Laboratories), sealed with nail polish, and imaged on a Leica DM5500B microscope with a 63×/1.4 NA objective at 1920 × 1440 resolution.

The antibodies used are as follows: Actin (Abcam ab8226), Gapdh (Biolegend 919501 and Covance MMS580S), HA (Sigma H6908 and Roche 11867423001), Tbc1d24 (Abcam ab101933 and Aviva ARP57438), MYC (Sigma M4439), Map2 (Millipore AB5622), Tau (Millipore MAB3420), NF200 (Sigma N4142), vGLUT1 (Synaptic System 135303), Homer1 (Synaptic System 160011), NeuN (Millipore ABN91), Ctip2 (Abcam ab18465), Cux1 (Abcam ab140042), cleaved caspase-3 (Cell Signaling Technology 9661) and Tbr1 (Abcam ab31946) and anti-mouse, anti-rat or anti-rabbit Alexa 594 or Alexa 488 for secondary antibodies (Life Technologies). All reagents for cell culture were from Gibco, unless otherwise specified.