Fig. 6.

Secretion of Cab₃₀₈Myc from transfected INS cells. (A) Scheme of the primary structure of Cab45 and the Cab₃₀₈Myc construct. (B) Cells were pulse labeled for 30 minutes with ³⁵S amino acids and chased continuously for 4 hours 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 panel) and anti-insulin (lower panel; proinsulin conversion intermediates highlighted with an asterisk). (C) Cells were labeled as in B and then either lysed immediately without chase (Time 0) or chased for 4.5 hours in the absence (−) or presence (+) of secretagogue (Stim). Media (M) were collected and cells lysed (C), and the samples were then analyzed by immunoprecipitation with anti-Myc followed by mock digestion (−) or endoglycosidase H (Endo H) digestion (+). By SDS-PAGE, Cab₃₀₈Myc migrates as two bands ranging from 33 to 35 kDa. (D) Double-labeled immunofluorescence distribution of Cab₃₀₈Myc in transfected INS-1 cells (using polyclonal rabbit anti-Myc, green) relative to the (Golgi) distribution of GM130 (in red).