Figure 10.

Characterization of BRAG2a-P956/957A and BRAG2a-ΔSec7 mutants. A–D, Synaptic targeting assays with the EGFP-tagged BRAG2a-P956/957A and BRAG2a-ΔSec7 mutants in cultured hippocampal neurons at DIV15. Representative immunofluorescence images of the dendritic shaft and spines of neurons transfected with EGFP-BRAG2a-P956/957A (A) and EGFP-BRAG2a-ΔSec7 (C). The morphology of dendrites and spines was visualized by coexpression of mCherry. Quantification of the ratio of EGFP fluorescence in spines versus dendritic shafts of EGFP-BRAG2a-P956/957A (B) and EGFP-BRAG2a-ΔSec7 (D). Note that both EGFP-BRAG2a-P956/957A and EGFP-BRAG2a-ΔSec7 were selectively targeted to synapses compared with mCherry. Quantification data for each group were obtained from 100 measurements from five transfected neurons. These results were confirmed by two independent experiments. E, Immunoprecipitation assays. HeLa cells expressing GluA2 and FLAG-BRAG2a-WT or FLAG-BRAG2a-ΔSec7 were immunoprecipitated with anti-BRAG2a, anti-GluA2 or normal rabbit IgG, and subjected to immunoblotting with anti-BRAG2a and anti-GluA2 antibodies. Note that BRAG2a-ΔSec7 failed to interact with GluA2. F, G, Arf6 activation assays. F, Representative immunoblots of Arf6 activation assays using GST-GGA3 from HeLa cells expressing Arf6-FLAG and FLAG-BRAG2a-WT or FLAG-BRAG2a-ΔSec7 mutant. G, Quantification of the normalized GTP-Arf6/total Arf6 ratio (G). Note that BRAG2a-ΔSec7 lacked the ability to activate Arf6. Data for each group were obtained from three culture plates (n = 3). These results were confirmed by three independent experiments. *p < 0.001 (t test). Scale bars, 2 μm.