Fig 5. Retinoschisin binding to ATP1B2 patch II mutants.

HEK293 cells were transfected with expression constructs for ATP1A3 in combination with expression constructs for different ATP1B2 variants mutated in patch II regions depicted within the figure. (A) 48 h after transfection, cells were subjected to recombinant retinoschisin for 1 h, followed by intensive washing. Cells transfected with expression constructs for only ATP1B2 served as a negative control, cells transfected with expression constructs for ATP1A3 and normal ATP1B2 served as a positive control in the retinoschisin binding assay [13]. Heterologous protein expression as well as retinoschisin binding was investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) 48 h after transfection, cell surface proteins were biotinylated, purified by streptavidin affinity chromatography and analysed by Western blotting using antibodies against ATP1B2. The ATP1A1 immunoblot was performed as loading control. (C) 48 h after transfection, cells were subjected to FACS analyses applying an anti-ATP1B2 antibody. Representative histograms of cells transfected with selected ATP1B2 patch II mutants are given in this figure. Light grey: histogram of untransfected HEK293 cells depicting unspecific background signals. Dark grey: histogram of transfected HEK293 cells.