Skip to main content
. 2011 Mar 24;491(3):181–186. doi: 10.1016/j.neulet.2011.01.032

Fig. 2.

Fig. 2

Expression and surface trafficking of a wild-type and non-SUMOylatable SEP-mGluR7 in HEK293 cells: (A) SEP-mGluR7 was transfected into HEK293 cells. Thirty-six hours post-transfection, surface proteins were labelled with biotin, the cells lysed and biotinylated surface proteins were isolated on streptavidin beads and subjected to Western blotting alongside total lysate for GFP (SEP). (B) SEP-mGluR7 was transfected into 13DIV primary cortical neurons and imaged for SEP expression 5 days later (upper panel). SEP fluorescence was reduced with a transient exposure to pH 6.0 buffer consistent with the majority of the signal arising from surface expressed SEP-mGluR7. Application of ammonium chloride, which collapses pH gradients across the plasma membrane and transiently equilibrates intracellular compartments to the extracellular pH increased fluorescence showing the total SEP signal (pH 7.4) or minimum signal (pH 6). The fluorescence of the region boxed in the upper panel is shown graphically in the lower panel. (C) SEP-mGluR7 is functional in HEK293 cells, as assayed for its ability to activate the ERK pathway. Data are the mean ± SEM, **p < 0.01 (Student's t-test, n = 3). (D) SEP-mGluR7 WT or K889R was transfected into HEK293 cells. Thirty-six hours post-transfection cells were lysed in SDS-PAGE loading buffer and subjected to Western blotting for GFP (mGluR7) or β-actin to ensure equal loading. (E) SEP-tagged mGluR7 WT or K889R was transfected into HEK293 cells. Thirty-six hours post-transfection, surface proteins were biotinylated and cells lysed. Equal amounts of protein were then incubated with streptavidin beads to isolate labelled surface proteins and subjected to Western blotting alongside total lysate for either GFP (mGluR7) or β-actin to confirm the specificity of surface labelling. The proportion of total mGluR7(K889R) at the surface is shown graphically normalized to the wild-type. The blots are representative of three separate experiments and the mean ± SEM are shown in the bar graphs; differences were not significant in Student's t-test.