Figure 5

Neuroligin-2 expression precedes that of KCC2. A, Representative immunoblot showing the time-course of NL2 and KCC2 expression in mouse cortex at postnatal day 1 (P1), P4, P8, P11, P15, and P20. Actin was used as loading control. B, Quantification of the NL2 and KCC2 expression level as measured by immunoblot (n = 3). Protein expression was normalized to the expression level at P20. C, Representative images showing the expression of HA-NL2 (green) and KCC2 (red) in mouse cortical neurons (14 DIV) transfected with HA-NL2. Empty arrowhead indicates KCC2 puncta without NL2 colocalization; Filled arrowhead indicates NL2 puncta without KCC2 colocalization; Arrow indicates colocalized NL2 and KCC2 puncta. Scale bar, 10 μm. D, Representative images showing the expression pattern of NL2 (green), KCC2 (red), and NL2 + KCC2 in HEK cells. Scale bar, 10 μm. E, Lack of co-immunoprecipitation revealed by HEK cells co-transfected with HA-NL2 and KCC2. Total protein lysate was immunoprecipitated with normal mouse IgG or mouse anti-HA antibody and immunoblotted with rabbit anti-KCC2 and anti-NL2 antibodies. F, No co-immunoprecipitation detected between NL2 and KCC2 in mouse brain cortical tissue. Total mouse cerebral cortex protein lysate was immunoprecipitated with normal rabbit IgG, rabbit anti-NL2, or rabbit anti-KCC2 antibodies. The precipitated proteins were then probed with rabbit anti-KCC2 or anti-NL2 antibodies to test whether co-immunoprecipitation occurred.