Figure 8.

CHO cell lines expressing either wild-type or mutant insulin 2. (a) Northern blot analysis of insulin. Total RNA (20 μg) from CHO (lane 1), CHO-Ins2^wt (clone w9; lane 2), and CHO-Ins2^Mody cells (clone a7; lane 3) were electrophoresed, transferred to a nylon membrane, and hybridized with a mouse Ins2 cDNA probe. (b) Insulin secretion of CHO-Ins2^wt (clone w9) and CHO-Ins2^Mody (clone a7). Both cells were seeded at a density of 2 × 10⁵ cells/6-cm dish. After 24 h, the cells were incubated with serum-free media for the indicated times. Insulin stored in the cells (black bars) and that released into the media (open bars) were measured using anti-insulin antibodies. Although cell density and time course were different, similar data were obtained from another independent experiment. (c) Proinsulin content and secretion. CHO-Ins2^wt (lanes 3 and 8 for clone w7; lanes 4 and 9 for clone w9), and CHO-Ins2^Mody cells (lanes 5 and 10 for clone a1; lanes 6 and 11 for clone a3; lanes 7 and 12 for clone a7) were incubated with serum-free media for 24 h. The cells (lanes 3–7) were then solubilized, and the media (lanes 8–12) were concentrated by 10% trichloroacetic acid. These samples were resolved with tricine–SDS-PAGE (16.5% polyacrylamide gel) in a reducing condition (100 mM DTT). Immunoblotting analysis was performed using anti–C-peptide antibodies. Lanes 1 and 2 contain human proinsulin standard and the islet protein from normal C57BL/6J mice, respectively. CHO, Chinese hamster ovary.