Figure 2
NEU1 is present at the plasma membrane in the absence of PPCA.
(A,B) Western blot on the plasma membrane proteins (biotinylated fraction) of (A) COS-7 cells co-transfected with NEU1-Flag and PPCA (1:2) and probed with a mouse monoclonal anti-Flag or -cathepsin A antibody, or (B) human macrophages, endogenously expressing both NEU1 and PPCA, and probed with a rabbit polyclonal anti-NEU1 and mouse monoclonal anti-cathepsin A antibody. A representative pattern of (A) three and (B) two experiments is shown. (C–E) Confocal images of (C) NEU1-Flag and integrin beta 1, or (D) NEU1-Flag and cathepsin A, or (E) NEU1-Flag and lysosome distribution in permeabilized COS-7 cells co-transfected with NEU1-Flag and PPCA (1:2). (F) Sialidase activity measured from adherent COS-7 cells co-transfected with NEU1-Flag, NEU1-HA, or HA-NEU1 and PPCA (1:2). Plates were incubated with 200 μM 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid substrate in MES 20 mM, pH 4.5. Sialidase activity was measured at 405 nm and results expressed as mean ± SEM of 3 independent experiments, each run in duplicate and normalized to the control (w/o); e.g. cells transfected with PPCA and empty vector. ***p < 0.001. (G) Western blot on the biotinylated fraction of COS-7 cells co-transfected with HA-NEU1 or NEU1-HA and PPCA (1:2), and probed with a rabbit monoclonal anti-HA antibody. A representative pattern of three experiments is shown. (H) Detection by flow cytometry of HA-NEU1 or NEU1-HA co-expressed in COS-7 cells with PPCA (1:2). Cells were permeabilized, or not, by 0.1% saponin and both constructs detected using a Dylight 488-conjugated mouse monoclonal anti-HA antibody. A representative pattern of three experiments is shown.
