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Figure 2. C99-GFP accumulates in the ERC after 
-secretase inhibitor treatment. A) In control cells C99-GFP (green) expression was below detection levels (a), because of constitutive -secretase-mediated processing. Transferrin (Tfn, red) labeling indicated properly formed ERCs in these cells (b and c, arrows). Treatment of the cells with DAPT (d–f) or L-685,458 (g–i) resulted in the stabilization of C99-GFP (d and g), and accumulation in the ERC together with transferrin (wide arrows in f and i, yellow), as well as in the plasma membrane (narrow arrows in f and i). All images are confocal. Scale Bars = 10 µm for a, b, d, e, g, h; 5 µm for c, f, i. B) Western blot analysis showed increased levels of C99-GFP protein in the -secretase inhibitor treated C99-GFP overexpressing cells as compared to those in the control cells. The blot was probed with anti-GFP antibody (top; 1:1000) to visualize C99-GFP levels, and reprobed with antiactin antibody (bottom; 1:1000) to verify equal loading of each lane. Arrows on the right indicate the C99-GFP and actin bands.
