Figure 8.

siRNA knockdown of ERp57 expression leads to increased extracellular TG2 activity. A, HUVECs were transfected with ERp57-specific siRNA for 48 h and lysed with RIPA buffer. Whole-cell lysates were blotted for total ERp57 expression. B, densitometric analysis of the Western blots shown in A (n = 3). β-Actin was used as the loading control and normalization factor. C, siRNA knockdown of ERp57 leads to a decrease in extracellular ERp57 expression. Images were taken in triplicate with three biological replicates (n = 9). Scale bar, 100 μm. D, quantitative analysis of extracellular ERp57 expression normalized to cell count. Extracellular ERp57 expression was significantly decreased (**, p < 0.01) compared with control siRNA-treated cells. E and F, extracellular TG2 activity is elevated in ERp57 siRNA-knockdown cells compared with control siRNA-treated cells (*, p < 0.05). TG2 activity is restored to basal levels upon addition of recombinant oxidized ERp57 (**, p < 0.01). 200 μm 5BP was added to cells for 3 h to visualize TG2 activity. Exogenous oxidized ERp57 was added 30 min prior to 5BP. During confocal microscopy analysis, at least three images were taken per replicate with three biological replicates per condition. A representative set of images are presented. All images can be found in Figs. S6–S8. 5BP incorporation was normalized to TG2 staining. Scale bar, 100 μm. All quantitative data are presented as average ± S.D., and statistical analyses were performed using Student's t test.