Fig. 4. Processing of proinsulin in 3T3L1 adipocytes. (A) Proinsulin and insulin separated by a peptide column. 3T3L1 adipocytes expressing the human insulin gene were labeled with ³⁵S-Met/Cys for 1 hour, then chased for 1 hour. Cells were disrupted, and cell lysates were immunoprecipitated with an anti-insulin antibody. The immunoprecipitant was separated using a superdex peptide column, and radioactivity in each fraction was counted. (B) Reverse phase HPLC analysis of authentic recombinant human insulin and bovine insulin. The authentic insulin standards (10 µg in 100 µl 3 M acetic acid) were applied to the HPLC system with an acetonitrile gradient and monitored by 210 nm. The elution times for human and bovine insulin are 21 and 19 minutes, respectively. (C) Mature insulin in 3T3L1 adipocytes cells detected by HPLC. 3T3L1 adipocytes expressing the human insulin gene were disrupted by sonication, then cell lysates were analyzed by HPLC. Each fraction from the HPLC was neutralized and assayed using a human insulin ELISA kit.

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