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. 2016 May 16;291(28):14620–14627. doi: 10.1074/jbc.M116.731026

FIGURE 3.

FIGURE 3.

Identification of INS/IL-1α motif. A, anti-IL-1α mAb ELISA detected pINSd but failed to recognize comINS, which is mature INS lacking the C-peptide. A commercial IL-1α ELISA kit did not recognize pINSd. B, pINSd was incubated for 60 min in the presence or absence of trypsin (Sigma-Aldrich). pINSd (100 ng/lane loaded) was properly detected as a band of ∼30 kDa, but pINSd was not observed where trypsin was added. C, the same batch of pINSd in the presence or absence of trypsin was examined for IL-6 induction in A549 cells. The production of IL-6 was found to be due to pINSd because trypsin abolished IL-6 production. D, sequence analysis of pINS and IL-1α revealed the INS/IL-1α motif. E, dot blot of recombinant pINSd and IL-1α protein with the mAb anti-IL-1α. Data in A are comparisons between a commercial IL-1α ELISA and IL-1α developed with the mAb anti-IL-1α. Data in C are comparisons between the presence and absence of trypsin. Data are mean ± S.E. (error bars). #, p < 0.001 from three replicates. Cont, control.