Figure 3.

R101Q mutation blocks NLGN4 glycosylation and retains it at intracellular compartments. A, Alanine substitution of NLGN4 N102 residue to prevent N-linked glycosylation (N-Gly). B, Western blot (left) illustrating mature (green arrowhead) versus immature (purple arrowhead) HA-NLGN4 products harvested from HEK293 cells expressing NLGN4 WT versus N102A mutant, with EGFP as a transfection control and GAPDH as a loading control. Summary graphs (right) of total, mature, and immature NLGN4 normalized to coexpressed EGFP levels. C, Example immunoblot (C) for NLGN4 WT and R101Q variant, before (untreated) and after enzymatic deglycosylation with glycosidases (i.e., Endo H and PNGase F). D, Bar graphs represent the relative positions of mature (left) and immature (right) NLGN4 products. To compare across multiple experimental batches with slightly variable mobility shifts, the position of all bands was normalized (dotted lines) to the corresponding distance between the mature and immature bands in the untreated R101Q condition. E, Representative images (left) of HEK293 cells expressing NLGN4 WT (top) or R101Q mutant (bottom), as visualized by an IRES-driven mOrange reporter, and stained with DAPI, HA antibody, and calnexin antibody (respective channels) under permeabilized conditions. Arrowheads point at intracellular HA-NLGN4 colocalized with soluble mOrange and ER marker calnexin. Summary graphs (right) quantify the correlation between HA-NLGN4 and calnexin signals, measured via Mander's coefficients. F, Same as E, except the cells were counterstained with GM130 antibody. The values on bar graphs represent the mean ± SEM for number of experimental batches (immunoblots) or fields of view analyzed (imaging)/number of independent batches. Statistical significance was weighed by two-tailed, paired (for immunoblots) or unpaired (for imaging), Student's t test. ***p < 0.005; **p < 0.01; *p < 0.05; ns, not significant (p > 0.05).