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. 2021 Aug 30;70(11):2580–2594. doi: 10.2337/db21-0422

Figure 1.

Figure 1

Insulin B-chain substitution R(B22)E. A: Schematic representation of insulin chains showing the respective disulfide bonds and the R(B22)E substitution. B: Transfection of Min6 cells with empty vector (EV), untagged WT human proinsulin, hPro-R(B22)E-CpepMyc, or hPro-C(A7)Y-CpepMyc (Akita-Myc). The media-bathing transfected cells were collected overnight, and both cell lysates and media were resolved by SDS-PAGE under reducing conditions and immunoblotting with anti-human-proinsulin or cyclophilin B (CypB) (loading control) (bottom panels). Cell lysates were similarly analyzed under nonreducing conditions to reveal disulfide-linked proinsulin complexes (top panel). C: Transfection of Min6 cells as in B (first four lanes) or cotransfection with both untagged WT human proinsulin and hPro-R(B22)E-CpepMyc (last lane). Proinsulin-R(B22)E is secreted from Min6 cells, and under these conditions, proinsulin-R(B22)E does not block the secretion of coexpressed untagged WT proinsulin.