Accumulation of C99 in response to MG132, CQ and lack of its cytosolic tyrosine residues.

<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its three tyrosine residues (bold underline). (B) Immunoblot analysis of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-3Y/A-F/P-D/A (C99-3Y/A). Cells were left untreated or treated with 1 µM DAPT for 16 h and subsequently analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin was used as loading control. The positions of molecular mass markers are indicated on the left. (C) H4 cells stably expressing C99-3Y/A were left untreated or treated for 16 h either with 1 µM MG132, 100 µM CQ, or with a combination of 1 µM MG132 and 100 µM CQ. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of C99-3Y/A left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bars represent the mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01.