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Sheep anti Human plasminogen antibody used for the detection of plasminogen in Jurkat cell lysates by western blotting.
Necrotic cells harbour misfolded proteins and generate surface-bound plasmin which increases their phagocytosis by human MoDCs. (A) Uninjured and necrotic Jurkat lymphocytes were stained with 10 mg/L 7AAD and 10 mg /L Thiazine Red for 15 min then subjected to flow cytometry. (B) The Triton-insoluble fractions of uninjured and necrotic Jurkat lymphocytes were subjected to SDS-PAGE under reducing conditions and subsequent Coomassie staining. (C) Uninjured and necrotic Jurkat lymphocytes were incubated with t-PA and/or plasminogen for 15 min and washed. Total cellular protein lysates were prepared and subjected to SDS-PAGE under reducing conditions and subsequent immunoblot analysis. Most of the t-PA used was in single-chain form (~70 kDa), however a small extent of two-chain t-PA was also apparent (the band indicated by the asterisk at ~35 kDa). (D) PKH67-labelled necrotic Jurkat lymphocytes were treated with the indicated reagents for 15 min, then washed and incubated with PKH26-labelled human MoDCs. 24 h later, the proportion of double-positive (PKH67positive and PKH26positive) MoDCs was assessed by flow cytometry. Data are displayed as fold-change in double-positive MoDCs (mean ± s.e.m.; n = 3–5 independent experiments). Data was normalized to the group where necrotic cells received no exogenous reagent. **p<0.01 and ***p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc analysis.
From: Borg RJ, Samson AL, Au AE-L, Scholzen A, Fuchsberger M, Kong YY, et al. (2015) Dendritic Cell-Mediated Phagocytosis but Not Immune Activation Is Enhanced by Plasmin.
PLoS ONE 10(7): e0131216.