FIGURE 5.

Whole-cell protein and cell-surface expression of GlyR α2 subunit variants. (A,C) Whole-cell protein and cell-surface protein fractions from HEK293 cells transfected with either wild-type GlyR α2 or GlyR α2 variants α2^V–22L, α2^N38K, α2^K213E, or α2^T269M were immunostained for GlyR α2 using the pan-GlyR α subunit antibody mAb4a (48 kDa). For cell-surface biotinylation assays, cells were also transfected with EGFP as an internal control to ensure that only cell-surface proteins (30 kDa) were isolated. Cadherin served as housekeeping protein for both whole-cell and cell-surface expression and was detected by a pan-cadherin (pan-CAD) antibody (130 kDa). (B,D) Quantification of whole-cell and cell-surface protein fractions, normalized to pan-cadherin. The expression of wild-type GlyR α2 subunit was set to 1 (reflecting 100%). We noted a significant reduction of cell-surface protein for all GlyR α2 variants compared to the wild-type GlyR α2 control; significance values are *p < 0.05, **p < 0.01. All results are detailed in Table 1.