Fig. 3. Loss of EHD1 in MEF cells and human fibroblasts causes accumulation of 1 integrins in internal vesicles. MEF cells were pulsed with 9EG7 monoclonal antibodies that preferentially recognize ligand-bound mouse 1 integrins for 1 hour at 37°C (A-H). The cells were then briefly acid-stripped to remove non-internalized antibody, and then either fixed/permeabilized (1-hour pulse; A,B) or chased for 1, 2 or 4 hours (C-H). At the end of every chase, cells were stripped again to remove any recycled 1 integrins from the plasma membrane and the remaining intracellular 1 integrins were detected by immunostaining with Alexa Fluor 568 anti-rat secondary antibody. Arrows depict accumulation of 1 integrins at the ERC in Ehd1^–/– MEFs (F). Levels of intracellular 1 integrins in Ehd1^+/+ MEF (G) and Ehd1^–/– MEF cells (H) were quantified (graph in I) by the LSM 5 Pascale software using the Profile function. Representative fields (comprising more than 100 cells) were profiled by measuring the mean fluorescence in the field (80 µm) every 4 µm, and obtaining a mean value for all sections sampled. Standard deviations are depicted in the error bars, and the Student's t-test values for significance calculated at P<0.0001. MEF cells were grown in culture, lysed, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblot analysis (J). Human fibroblast cells growing on culture dishes (K) or coverslips (L,M) were treated with mock-RNAi or RNAi specific for EHD1 for 48 hours. Efficacy of RNAi was confirmed by immunoblotting with anti-EHD1 (K; top panel) and anti-actin as a control for equal protein loading (K; bottom panel). Mock-treated (L) and EHD1-RNAi-treated (M) human fibroblasts on coverslips were incubated with anti-human 1 integrin antibodies (MCA2028; Serotec) for 2 hours, briefly acid-stripped and chased with complete media for 2 hours. Intracellular 1 integrins were visualized by confocal microscopy following incubation with Alexa Fluor 568 anti-mouse secondary antibody under permeabilizing conditions. The micrographs shown are from a representative experiment from four independent experiments. Bars, 10 µm.