Fig. 3.

Survey of substitutions at position B24 of human proinsulin. A secretion screen was performed in HEK293T cells following transient transfection of plasmids encoding WT human proinsulin, B24-variant proinsulins, or an empty vector (EV). The cells were metabolically labeled with ³⁵S-Met/Cys for 30 min followed by 90-min chase. At the end of the chase, media (“M”) were collected and cells (“C”) lysed. Cell lysates and media were immunoprecipitated with antiinsulin. Immunoprecipitates were analyzed with NuPage (Invitrogen) under reducing conditions. B24 substitutions are indicated above corresponding lanes at Top; the empty vector and WT human proinsulin are shown in each of the four gels. A–D Represent separate gels with vertical blue lines separating nonconsecutive lanes from the same gel; complete gels are shown in SI Appendix, Fig. S3.