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Fig. 3. ARF6 activation upon infection. HeLa cells in suspension were centrifuged for 20 seconds with or without bacteria (control) and incubated for the indicated times at 37°C as described in the Methods section. The cells were then lysed on ice and incubated with the ARF binding domain of ARHGAP 10 (ARF-BD) fused to GST, which binds the active GTP-bound form of ARF6. The proteins associated with this GST construct were pulled down using glutathione sepharose and analysed by western blot using anti-ARF6 antibodies. Aliquots of total cell lysates were immunoblotted for total ARF6, showing that the total amount of protein was identical at all time points. Data are representative of five experiments.
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