Fig. 1

Production and characterization of antibodies against human NLGN4. A The domain structure of human NLGN4. The positions of immunogens used to produce anti-NLGN4 antibodies are shown at the bottom. B Anti-NL4-p1 antibodies specifically react with NLGN4 but not with the other NLGNs in Western blotting. Lysates of HEK293 cells overexpressing FLAG-tagged NLGN1 (FLAG-NL1), NLGN2 (FLAG-NL2), NLGN3 (FLAG-NL3), or NLGN4 (FLAG-NL4) were electrophoresed and immunoblotted with anti-FLAG and anti-NL4-p1 antibodies. Anti-α-tubulin antibodies were used as a loading control. Anti-NL4-p1 antibodies reacted with FLAG-NL4 and did not cross-react with FLAG-NL1, 2, and 3. Lysates of HEK293 cells transfected with empty FLAG vector or pcDNA3.1 were used as a negative control (mock). C Anti-NL4-p1 antibodies specifically react with NLGN4 but not with other NLGNs in fluorescent immunocytochemistry. HEK293 cells expressing FLAG-NL1, 2, 3, or 4 were immunolabeled with anti-FLAG (green) and anti-NL4-p1 (red) antibodies. Merged images with DAPI nuclear staining (blue) are shown at the bottom. Anti-FLAG antibodies recognized FLAG-NL1, 2, 3, and 4 expressed on the cell membrane. Anti-NL4-p1 antibodies recognized only FLAG-NL4 but not FLAG-NL1, 2, and 3. Scale bar = 10 μm. D Anti-NL4-p1 and anti-NL4-m1 antibodies react with endogenous NLGN4 expressed in human iPSCs. Cell lysates from human iPSC clone 610B1 (hiPSC WT) and its NLGN4X knockout derivative (hiPSC NL4-KO) were electrophoresed and immunoblotted with anti-NL4-p1, anti-NL4-m1, anti-NLGN4 (GTX 116674), and anti-β-actin antibodies. Endogenous NLGN4 proteins in original hiPSC WT lysates were detected with anti-NL4-p1, anti-NL4-m1, and GTX 116674 antibodies, whereas no signals were detected in hiPSC NL4-KO lysates