Figure 5. Secretory rescue and stabilization of mutant proinsulin or Tg by their WT counterparts is linked to the WT/mutant expression ratio.

(A–D) 293T cells were transiently cotransfected with the indicated plasmid combinations, with empty vector added to keep total DNA per well constant. (A) Total mutant proinsulin recovery at 20 hours after synthesis was measured by pulse chase (see Methods). Protein stability was quantified by band recovery at 20 hours chase relative to that at time 0 (mean ± SEM, n = 4, *P < 0.05). (B) Overnight media and cell lysates were analyzed by immunoblotting with anti-GFP (normalized to total cellular protein). Representative blots from 3 experiments are shown. (C) Overnight media were collected, and hPro secretion (normalized to total cellular protein) was measured by RIA. Data represent mean ± range from 2 independent experiments. (D) Overnight media and cell lysates (bottom 2 rows) or combined lysate and media (top row) were analyzed by immunoblotting with anti-GFP (normalized to total cellular protein). Representative blots from 3 experiments are shown. (E) INS1E cells were transfected with 2 or 0.5 μg plasmid expressing mutant proinsulin; the cellular levels of hProG(B23)V-CpepSfGFP are shown at left. Cell lysates and basal secretion (B) and glucose-stimulated secretion (S), normalized for hProG(B23)V-CpepSfGFP protein expression (1% of total for 2 μg transfection; 3% of total for 0.5 μg transfection), were analyzed by immunoblotting with anti-GFP. Percentage secretion was quantified as total GFP signal in media over total in cells. The data represent mean ± SEM, from 3 independent experiments. *P < 0.05.