Cells were plated into six-well plates 1 day before transfection. A total of 2 μg of plasmid DNA was transfected using Lipofectamine 2000 (Invitrogen). For metabolic labeling, cells were pulse-labeled with 35S-Met/Cys for 15 min, washed, and chased with label-free media for 0, 60, or 120 min as indicated. Immediately thereafter, media and cell lysate were immunoprecipitated with anti-insulin or anti-AAT antibodies, and immunoprecipitates were subjected to 4 to 12% gradient SDS-PAGE under reducing conditions. Preproinsulin, proinsulin, insulin, and AAT were analyzed using a Typhoon Phosphorimager (GE Healthcare). Band intensities were quantified using National Institutes of Health ImageJ. For selective plasma membrane permeabilization and Proteinase K digestion, cells were incubated on ice with phosphate-buffered saline (PBS), with or without Proteinase K (10 μg/ml), 0.01% digitonin, or 1% Triton X-100 as indicated for 30 min. Cells were harvested, boiled in SDS sample buffer, and analyzed by immunoblotting with anti-proinsulin antibodies.

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