Cells were serum-starved for 2 hours at 37°C in DMEM/0.5% BSA and stained with unlabeled mAbs detecting either murine β1 integrin (BD Biosciences, clone 9EG7) or human β1 integrin (Abcam, clone 12G10) at 5 μg/ml in DMEM/0.5% BSA on ice for 30 min (surface binding). Cells were transferred to 37°C for 1 hour to allow internalization (pulse). After pulse, cells were briefly acid-rinsed in 0.5% acetic acid and 0.5 M NaCl (pH 3.0) for 45 s to remove noninternalized and surface-bound antibodies. Cells for pulse-only conditions were either fixed (surface β1 integrin) or fixed and permeabilized (internalized β1 integrin). Cells undergoing chase were chased in antibody-free complete medium for 2 or 4 hours at 37°C to assess receptor recycling back to the surface (Chase/2h or Chase/4h), and cells were fixed or fixed/permeabilized. The remaining β1 integrin mAbs in pulse-only and pulse/chase conditions were detected by with an FITC-labeled secondary antibody and measured by IF or flow cytometry. Cells were exposed to 25 μM of the myristoylated ARF6 2-13 peptide or vehicle before assay.

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