Fig. 2.

Ribosome stalling during co-translational ER translocation of APP.C99. a WB analysis of APP.C99 proteins from fly muscle expressing an APP.C99 transgene with a Myc tag at the C-terminus, using the N-term 6E10 and C-term Myc antibodies. b WB analysis of APP.C99 proteins from HeLa cell expressing APP.C99, using the N-term 6E10 and C-term C1/6.1 antibodies. c WB analysis of APP.C99 proteins from HeLa cell expressing ER-Flag-C99-Myc, using the N-term 6E10 and C-term Myc antibodies. d Pum labeling of stalled APP.C99 NPCs. Stalled NPCs were labelled with emetine plus Pum after HHT treatment to let actively translating ribosomes to run off. Pum-labelled protein were immunoprecipitated with anti-Pum and the presence of APP.C99 was detected with 6E10. The ER protein Calnexin serves as negative control. e Immunostaining of APP.C99 transfected HeLa cells with the amyloid conformation-specific mOC78 antibody and anti-Pum, which labeled stalled NPCs after active ribosomes were let run off by HHT treatment. Scale bar, 5 µm. f Immunostaining of APP.C99 transfected HeLa cells with 6E10 and mOC78 antibodies in control and anisomycin treatment conditions. Scale bar, 10 µm. g Immunostaining of APP.C99 transfected HeLa cells with mOC78 and Pum antibodies in control and anisomycin treatment conditions. Scale bar, 5 µm. h Co-IP assay in untransfected (UNT) and APP.C99 transfected HeLa cells. Lysates were immunoprecipitated with anti-RPL22 or anti-Sec61B antibodies, and the immunocomplexes were probed with the indicated antibodies. Immunoblots represent at least 3 independent repeats