Figure 7.

mAb binding specificities. A, mAb binding to ZnT8-His immobilized to Ni-NTA plate. Bound mAb20 (red) or mAb39 (magenta) was detected by an HRP-conjugated secondary antibody. Solid lines, least-squares fits of binding data points to a one-component binding process. Error bars, S.E. (n = 8). B, mouse ZnT8-His (M) and human ZnT8-His (H) were SDS-denatured on immunoblots and detected by mAb20 or mAb39, as indicated. A replicated immunoblot was probed by an anti-His tag antibody to show that approximately the same amounts of HEK293 crude lysates were loaded to each lane. All immunoblots were recorded using an identical exposure time on Amersham Biosciences Imager 600. C, detection of endogenous human ZnT8 in EndoC-βH cells by mAb20 or mAb39 immunoblotting. An equal amount of EndoC-βH lysate (∼50 μg of total protein) was loaded to each lane. D, immunoblotting of HEK lysates with overexpression of a ZnT homolog as indicated. An approximately equal amount of total lysate protein (5 μg) was located to each lane, probed by either mAb20, mAb39, or anti-FLAG antibody, as indicated. Note that the mAb39 immunoblot was recorded with an over 100-fold longer exposure time as compared with that of the mAb20 immunoblot. a.u., arbitrary units.