Biochemical and immunostaining studies of TMD0 harboring select R74X or E128X mutations. (A and C) Representative immunoblots of FLAG-tagged SUR1 TMD0 constructs with either R74X (A) or E128X (C) mutations expressed in COSm6 cells. Blots were probed for fTMD0 (α-FLAG) followed by α-tubulin as a loading control. (B and D) Densitometry analysis was performed on the blots in A and C to quantify R74X (B) or E128X (D) fTMD0 protein levels relative to WT fTMD0. n = 3. (E) Cells cotransfected with WT, R74W, R74K, or E128K fTMD0 and Kir6.2ΔC36 were probed with α-FLAG antibody 48 h after transfection to detect surface expression of mini-K_ATP channels. A rim of surface staining was detected in WT- and E128K-transfected cells (green, inset images), but not in either R74W- or R74K-transfected cells. Cell nuclei were DAPI stained (blue). Bar: 50 mm; insets, 10 mm.