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. 2015 Aug 31;290(41):25188–25196. doi: 10.1074/jbc.M115.667915

FIGURE 2.

FIGURE 2.

Increased IL-1α activity in activated ECs occurs due to IL-1α cleavage by calpain. A and B. level of IL-1α transcript relative to GusB by quantitative PCR (A) or protein by Western blot in whole cell lysates of HUVECs pretreated as indicated. C, mean fluorescent intensities (MFI) of HUVEC cells, pretreated as indicated, stained for IL-1α, and analyzed by flow cytometry. D, relative level of calpain activity using a substrate assay in necrotic lysates of the cell types treated as indicated. E, ELISA analysis of recombinant mature (p17) and pro-IL-1α (p33) at the concentrations indicated (pg/ml) and endogenous IL-1α within necrotic VSMC lysates made with and without calpain inhibition (+Ci). F, Western blot for IL-1α showing calpeptin (Ci) prevents processing of p33 to p17. G, mature IL-1α (p17) content by ELISA of necrotic HUVEC lysates pretreated as indicated and made with and without calpain inhibition (+Ci). Data represent the mean ± S.E. of n = 2 (A and C), 3 (E), and 4 (F); *, p ≤ 0.05, **, p ≤ 0.01; NS = not significant.