Figure 1

Knockdown of neuroligin-2 decreases KCC2 expression. A, Efficient knockdown of NL2 by NL2shRNA or NLmiR in mouse cortical neurons. Neurons were co-transfected with mCherry at 2 DIV and HA-NL2 expression was assayed by HA-immunostaining at 8 DIV. B, shRNA-proof mutant HA-NL2* was resistant to the knockdown of NL2shRNA or NLmiR. Scale bar, 10 μm. C, Bar graphs showing quantified somatic HA-immunostaining intensity (normalized by mCherry controls). ***p < 0.001 (one-way ANOVA). D, Representative images showing reduced KCC2 immunostaining (green) in NLmiR-transfected neurons (arrowhead) compared to non-transfected neurons (arrow, 12 DIV). Scale bar, 20 μm. E, NL2*, but not NL1* rescued the KCC2 level when coexpressed with NLmiR. Inlets showed HA-immunostaining (gray) to confirm the expression of HA-NL1* and HA-NL2*. Scale bar, 20 μm. F, NL2shRNA also decreased KCC2 expression level. Scale bar, 20 μm. G, Bar graphs showing quantified somatic KCC2 immunofluorescence intensity in non-transfected (nonTF) neurons (173 ± 4 a.u.) and neurons transfected with mCherry (172 ± 10), NLmiR (73 ± 8), NLmiR + NL1* (93 ± 9), NLmiR + NL2* (120 ± 12), or NL2shRNA (80 ± 11). a.u., arbitrary unit. **p < 0.01, ***p < 0.001, n.s., not significant, one-way ANOVA. H, NL2shRNA specifically knock down NL2 but not KCC2 in HEK 293T cells, which were co-transfected with NL2 + GFP, NL2 + NL2shRNA, KCC2 + GFP, or KCC2 + NL2shRNA. Total protein lysate was analyzed by immunoblot. Actin was used as loading control. I, NKCC1 immunostaining signal (red) was not altered in NL2shRNA-transfected neurons (green, arrowhead, 9 DIV). Scale bar, 20 μm. Bar graphs show the quantified somatic NKCC1 signal intensity (p > 0.7, unpaired Student’s t-test).