Figure 1
Plasma membrane and cytosolic IDE have different turn-over. A-Immunofluorescence of PM-associated endogenous IDE in living N2a wild type (N2WT) (panel 1) and Na2WT-EGFP (panel 2) cells using 1C1 monoclonal antibody and Alexa 488 (green) or Cy3 (red) anti-mouse IgG, respectively. Expression of EGFP was visualized based on the EGFP fluorescence (green) and labeled the cytosolic compartment. Scale bars: 5 μm. B-Immunogold electron microscopy on cryosections of N2aSW cells showed a cluster of IDE molecules at the PM (panel 1) which were more evident at higher magnification of the framed region (panel 2). Scale bar, 50 nm. C- Representative phosphorimaging of the remaining IDE during the chase period in cytosol (upper panel) and PM (lower panel). N2aWT cells were pulse-labeled with [35S]-methionine for 30 minutes and chased for the indicated times (0 to 96 hours). Cytosolic and PM fractions were immunoprecipitated as described in Material and Methods. The intensity of the bands was quantified and the maximal value was obtained at time cero and after 6 hours of chase for cytosolic and PM-associated IDE pools, respectively.