Figure 2.
The clustering of the γ2-GABAA receptor on the neuronal surface is specifically induced by Wnt-5a. A, Neurons (18 DIV) were treated with control media, Wnt-3a or with Wnt-5a ligand for 15 min, or coincubating with Wnt-5a/sFRP. Then neurons were labeled with Sulfo-NHS-LC-Biotin and processed at 4°C. The GABAA-Rs present in neuronal surface and total protein was analyzed using SDS-PAGE and Western blot with the antibody against the γ2 subunit. Transferrin (TfR), another receptor, was also detected as control. B, Quantification of the blots (n = 3). C, GABAA-Rs were immunostained with antibody to γ2 subunits in nonpermeabilized cells showing clustering of the receptor. Representative images of control hippocampal neurons or those treated with Wnt-5a or Foxy-5, at the time indicated, are shown. D, The number of clusters in 20 μm of neurite (Da) and the size (μm2, normalized to the control) (Db) were analyzed (n = 5). Error bars indicate SEM. *p < 0.05.