Calreticulin affects the production of Aβ₄₀ and Aβ₄₂.

<p>(<b>A, B</b>) CHO cells overexpressing APP were mock-transfected (mock) or transfected with constructs encoding the P-domain (P), the N-domain (N) or the N- and the P-domain (NP) of calreticulin or full-length calreticulin (CRT). (<b>B</b>) Transfected cells were subjected to cell surface biotinylation, biotinylated proteins were isolated and Western blot analysis of cell lysates and biotinylated cell surface proteins with the APP antibody A8717 was performed. (<b>C</b>) CHO cells overexpressing APP were incubated with GST-calreticulin (CRT), GST/C-domain (C) or GST/P-domain (P). (<b>D</b>) Amino acids 706–710 of APP comprising the γ-cleavage site for generation of Aβ₄₀ and Aβ₄₂ the sequence of the corresponding antisense peptide and the inverted sequence of the antisense peptide are shown. Similarities between the inverse antisense peptide sequence and amino acid 330–344 of calreticulin are indicated (bar, colon and dot represent identical, highly conserved and weakly conserved amino acids). NT: N-terminus; CT: C-terminus. (<b>E</b>) Label-free binding assay using substrate-coated Aβ₄₀ or Aβ₄₂ peptides and soluble calreticulin peptide comprising amino acids 330–344 (CRT pep) or antisense peptide (anti pep). Mean values ± SD from triplicates of a representative experiment are shown. (<b>F</b>) GST/C-domain fusion was incubated with a detergent extract of mouse brain homogenate in the absence (-) or presence of calreticulin peptide (CRT pep) or antisense peptide (anti pep). Proteins bound to the GST/C-domain were precipitated with glutathione-agarose (pull-down) and subjected to Western blot analysis with the APP antibody A8967. Aliquots taken from the samples before precipitation were used as input control. (<b>G</b>) CHO cells overexpressing APP were incubated in the absence or presence of the calreticulin peptide (CRT pep) or antisense peptide (anti pep). (<b>A, C, G</b>) Cell culture supernatants were collected and the amounts of Aβ₄₀ and Aβ₄₂ in cell culture supernatants were determined by ELISA. The levels obtained after mock-transfection were set to 100%. Mean values ±SEM from 3 independent experiments with biological duplicates per experiment are shown (Students t-test * p>0.05, ** p>0.005).