Mab1598

Cell cultures were fixed in 4% PFA in PBS solution for 8 min and washed 3 × 6 min in PBS. Next, cells were permeabilized in 0.1% Triton X-100 (Bioshop, TRX506) in PBS solution for 10 min in room temperature (RT) and blocked with 10% NDS in PBS solution for 1 hour in RT. Cells were incubated with primary antibody solution with 5% NDS, rabbit anti-mCherry antibody (1:500; Abcam, ab167453, RRID:AB_2571870) and mouse anti-PSD-95 antibody (1:500; Merck-Millipore, MAB1598, RRID:AB_94278) in PBS overnight in 4 °C. Next, the cells were washed 3 × 10 min and incubated with secondary antibody solution: 5% NDS, anti-rabbit Alexa Fluor 647 (mCherry coding plasmids; 1:500; Invitrogen, A31573, RRID:AB_2536183) and anti-mouse Alexa Fluor 488 (mCherry coding plasmids; 1:500; Invitrogen, A21202, RRID:AB_141607) or anti-mouse Alexa Fluor 555 (GFP coding plasmids; 1:500; Invitrogen; A31570; RRID:AB_2536180) in PBS for 1h in RT. Then the cells were washed 3 × 10 min and mounted on microscope slides with Fluoromount G with DAPI (Invitrogen, 00-4959-52).

Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Information of primary antibodies is as follows: mouse anti-FLAG (Sigma, F1804, 1:5000 for WB), mouse anti-GFP (Santa Cruz, sc-9996, 1:1000 for WB), mouse anti-GFP (Invitrogen, A-11,120, 1:1000 for staining), mouse anti-PSD95 (Millipore, MAB1598, 1:1000 for WB and 1:500 for staining), mouse anti-synaptophysin (Dako, M7315, 1:5000 for WB), rabbit anti-β-actin (Santa Cruz, sc-1616-R, 1:1000 for WB), mouse anti-Tau-1 antibody (Millipore, MAB3420, 1:500 for staining), mouse anti-MAP2 antibody (Millipore, MAB3418, 1:500 for staining), DGCR2 antibody was generated against hDGCR2-ICD in rabbit (1:1000 for WB and 1:200 for staining).
To generate FLAG-hDGCR2, the human DGCR2 cDNA encoding 22–550 amino acids of DGCR2 without signal peptide was amplified by PCR and subcloned into pFLAG-CMV1 (Sigma, E7273) downstream of an artificial signal peptide sequence and a FLAG epitope. Different cDNAs encoding ΔECD and ΔICD of DGCR2 were amplified with primers 5’- GAAGATCTGATGCGCCTGGTCGTC-3’ and 5’- ACGCGTCGACCTACACCACAGTATTG-3’, 5’-GAAGATCTGCGGCCAGAGCTG-3’ and 5’- ACGCGTCGACCTACCGGTGGACCATGAAG-3’, and subcloned into pFLAG-CMV1 separately. NRXNs and NLGNs constructs were obtained as described previously [48 (link)]. The authenticity of all constructs was verified by DNA sequencing and western blotting analysis.

The following antibodies were used for western blot: Anti-neuroserpin goat polyclonal antibody G64 (generation and affinity-purification have been previously described^{13 (link)}) (0.5 ug/ml); Anti-synaptophysin (Abcam ab32594, 1:1000); Anti-SNAP25 (Abcam ab41455, 1:1000); Anti-neuroserpin (Abcam ab46761, 1:5000 and ab32901, 1:2000); Anti-synapsin-I (Cell Signaling Technology D12G5, 1:1000); Anti-PSD-95 (Millipore clone EP2652Y, 1:1000); Anti-beta-actin (Millipore clone C4, 1:5000); Anti-PDI (StressMarq SPC114C, 1:2000); Anti-GFP (Clontech mouse Living Colors monoclonal antibody 632,459, 1:5000). The following antibodies were used for immunofluorescence: Anti-MAP2 (Sigma M4403, 1:200); Anti-cleaved caspase 3 (R&D Systems AF835, 1:100); Anti-synaptophysin (Abcam ab32594, 1:200); Anti-PSD-95 (Millipore MAB1598, 1:100); Anti-neuroserpin goat polyclonal antibody G64 (3 ug/ml).

Proteins from brain tissue lysates or primary cortical neurons were extracted using RIPA buffer with protease and phosphatase inhibitor cocktails (Roche). Total lysates were obtained by 30 s of centrifugation at 4 °C. The protein concentration of the lysates was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Then, 10–50 μg of proteins were loaded to SDS-PAGE gels and transferred and transferred to nitrocellulose membranes for 1 h. Membranes were blocked with Tris-buffered solution with 0.1% tween, 5% skimmed milk, and 2% of FBS for 1 h at room temperature (RT) and incubated with PSD95 (1:1.000, MAB1598; Millipore), PrP (1:500; 6H4; Thermo) or Actin (1:20.000; MAB1501; Millipore) antibodies at 4 °C O/N. Following HRP-linked secondary antibody (Dako) incubation for 1 h at RT, membranes were developed with ECL substrate (Thermo).