Dominant-negative blockade of proinsulin-WT caused by preproinsulin-A(SP24)D, miscleaved proinsulin, and uncleaved preproinsulin. Dominant-negative blockade.

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Dominant-negative blockade of proinsulin-WT caused by preproinsulin-A(SP24)D, miscleaved proinsulin, and uncleaved preproinsulin. Dominant-negative blockade of proinsulin-WT caused by preproinsulin-A(SP24)D, miscleaved proinsulin, and uncleaved preproinsulin. A: 293T cells were cotransfected to express human preproins-WT with either mouse preproins-WT, A(SP24)D, or A(SP24)D-DelCys [in which all cysteines of A(SP24)D were mutated (21)] at a DNA ratio of 1:2. Beginning at 30 h posttransfection, cells were incubated with DMEM containing 25.5 mmol/L glucose plus 10% FBS for additional 16 h. Media were collected, and a human proinsulin-specific RIA was used to measure secretion of coexpressed human proinsulin-WT. The secretion of human proinsulin coexpressed with mouse preproins-WT served as a positive control (i.e., set to 100%). Results are shown as mean ± SD from three independent experiments. B: INS1 cells cotransfected with preProCpepGFP-WT with either preProCpepMyc-WT or A(SP24)D (at a DNA molar ratio of 1:2) were labeled for either 0.5 or 20 h as indicated. The cell lysates (C) and media (M) were immunoprecipitated with anti-GFP and analyzed under reducing conditions. C: INS1 cells transfected to express preProCpepMyc-WT, A(SP24)D, or PFD were lysed at 48 h posttransfection. The cell lysates were resolved by 4–12% NuPage under reducing conditions. The proteins were electrotransferred to nitrocellulose and immunoblotted with anti-Myc antibody. D: 293T cells cotransfected with preProCpepMyc-WT and untagged AAAAD-Proins (processed to AD-Proins; see Fig. 5A) at a range of DNA ratios (indicated below) were pulse-labeled with 35S-Cys/Met for 30 min and chased for 0 or 2 h. Cell lysates (C) and collected chase media (M) were immunoprecipitated with anti-insulin and analyzed under reducing conditions. E: 293T cells cotransfected with untagged preproinsulin-WT (processed to Proins) and preProCpepMyc-WT, A(SP24)D, or PFD were pulse-labeled with 35S-Cys/Met for 30 min and chased for 0 or 3 h. Cell lysates (C) and collected chase media (M) were analyzed as in D. Ming Liu et al. Diabetes 2012;61:828-837 ©2012 by American Diabetes Association