Fig. 3

Patient serum does not compete with mAb2b for the same binding sites at the human GlyR?1 subunit. (A?C) Left images: Incubation schemes of Patient 1 (pat1) serum and mAb2b incubated either together for 2?hours or successively for 1?hour each. (A?C) Middle images: Fluorescence signals of GlyR?1hs transfected HEK293 cells incubated with pat1 serum (1:50) and mAb2b (1:50) using 3 different protocols. (A?C) Right images: Different antibody concentrations were used (pat1 serum 1:50 and mAb2b 1:2,000). (D?F) Fluorescence signals of GlyR?1hs transfected HEK293 cells incubated with pat1 serum (1:50) and mAb2b (1:100; 1:500; 1:1,000) together for 2?hours (D), successively first with pat1 serum for 1?hour followed by 1?hour with mAb2b (E), and successively first 1?hour with mAb2b followed by pat1 serum for 1?hour (F). (G) Cells were incubated with pat1 serum 1:50 twice. Following the first pat1 incubation, labeled protein was stained with secondary goat antihuman Cy3 (left). Pat1 serum was applied again for 1?hour and stained with goat antihuman Alexa Fluor 488 (middle). (H) Cells were incubated with mAb2b 1:500 followed by secondary staining with goat antimouse Cy3. mAb2b was applied again for 1?hour and stained with goat antimouse Alexa Fluor 488. Scale bars = 10?m.