Regulation of Amyloid Precursor Protein Processing by the Beclin 1 Complex (original) (raw)
Figure 5
Inhibition of autophagosomal turnover leads to a reduction in Becn1 and Pik3c3 levels.
A–E. B103/hAPP cells were treated with vehicle (DMSO) or 50 nM BafA for 24 hrs to inhibit autophagosomal degradation. Western blots (A) and quantification (B–E) of RIPA cell lysates that were probed with CT15 APP, LC3, Becn1, and Pik3c3 antibody. An actin antibody was used as a loading control. F. Confocal microscopy of B103/hAPP cells treated with vehicle (DMSO) or 100 nM BafA for 24 hrs. Cells were stained with CT20 APP antibody (magenta) and LC3 antibody (cyan). Co-localization is indicated in yellow. Arrowheads indicate LC3 positive APP containing vesicles. The arrow indicates an APP containing LC3 negative vesicle (scale bar represents 10 µm). The line indicates cross-section. Cyan line in the cross-section represents APP intensity, magenta line represents LC3 intensity (AU). G–L. CHO/hAPP cells were treated with vehicle (DMSO), 50 nM, or 100 nM BafA (WB data not shown) for 24 hrs. Western blots (G) and quantification (H–L) of RIPA cell lysates that were probed with the CT15 APP, LC3, Becn1, and Pik3c3 antibody. An actin antibody was used as a loading control. M–N. BafA and CQ treatment cause increased APP processing which in turn leads to elevated levels of secreted APP (sAPP) in the cell supernatant (M). This is quantified in (N). O. Epifluorescence microscopy of CHO/hAPP cells treated with vehicle (DMSO) or 100 nM BafA for 12 hrs. Cells were stained with the 8E5 APP antibody (magenta) and LysoTracker (cyan). Co-localization is indicated in yellow (scale bar represents 25 µm). P. Schematic representation of the APP antibody epitopes. Bars are mean ± SEM from triplicate cultures of at least two independent experiments. * p<0.05, ** p<0.01, *** p<0.001 by unpaired Student's t test.