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. 2012 Mar 8;7(3):e33350. doi: 10.1371/journal.pone.0033350

Figure 4. Integrin expression regulates ADAM17 sheddase activity.

Figure 4

(A) Time-dependent release of alkaline phosphatase (AP)-tagged HB-EGF by unstimulated (C) and 5-HT stimulated cells. Cells were transfected with AP-HB-EGF expressing plasmid and 2 days after transfection they were stimulated with 5-HT for the indicated time. AP activity of cell supernatants was determined using Attophos substrate. Data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units; *p<0.05, **p<0.01 vs control at same time point, n = eight experiments, three parallels/each condition. (B) β1 integrin silencing promotes 5-HT-induced AP-HB-EGF shedding. Cells were transfected with AP-HB-EGF expression plasmid together with β1 integrin siRNA (β1-siRNA) or a non-targeting (nt−) siRNA. Successful silencing of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. (C) β1 integrin overexpression inhibits 5-HT induced AP-HB-EGF release in mesangial cells. Cells were transfected with AP-HB-EGF and with β1 integrin expressing plasmid (β1-plasmid) or control DNA (Co-DNA). Successful overexpression of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. Activity data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units (RFU); *p<0.05, **p<0.01 vs unstimulated control nt-siRNA or control DNA-transfected cells; # p<0.05 and ## p<0.01 vs 5-HT stimulated nt-siRNA or DNA-transfected cells; n = five experiments, three parallels/each condition.