Fig. 8. Short-term secretion of newly synthesized Cab₃₀₈Myc and AAT from untransfected or transfected INS-832/13 cells. (A) Cells were pulse labeled for 30 minutes with ³⁵S amino acids and chased for 40 minutes in the absence (-) or presence (+) of secretagogue (Stim). Media (M) were collected and cells lysed (C), and the samples were then analyzed by sequential immunoprecipitation with anti-Myc (upper panels) and anti-insulin (middle panels), or anti-AAT (lower panels). A representative clone transfected with the Cab₃₀₈Myc construct (Clone 2) is shown on the right. Untransfected INS-832/13 cells (left) serve as a negative control for the anti-Myc and anti-AAT immunoprecipitations. The positions of proinsulin (Proins) and insulin are shown; higher bands represent proinsulin conversion intermediates. (B) Two additional independent clones transfected with the Cab₃₀₈Myc construct (Clones 34 and 24) are shown with less Cab₃₀₈Myc expression. The experimental protocol was identical to A except that cells were pulse labeled for 40 minutes. For clarity, lines have been added at bottom indicating the stimulus-dependent secretion of newly synthesized insulin. As shown, stimulus-dependent secretion of Cab₃₀₈Myc was zero in these clones. These experiments have been repeated and confirmed.