Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

Figure 2

Rab35 is inactivated during phagocytosis of zymosan. (a) GTP-dependent association of GST-ANKR with Rab35-Q67L. COS-7 cell lysates expressing GFP-Rab35-Q67L or GFP-Rab35-S22N were incubated with GST or GST-ANKR. The proteins associated with GST or GST-ANKR were pulled down using glutathione-sepharose beads and analyzed by western blotting (top panel). The middle panel shows aliquots of total cell lysates. (b) Binding specificity of GST-ANKR. Glutathione-sepharose beads coupled with GST-ANKR were incubated with COS-7 cells lysates expressing GFP-Rab35-Q67L, GFP-Rab21-Q78L, or GFP-Rab11-Q70L. Proteins bound to the beads were analyzed by western blotting. The middle panel shows aliquots of total cell lysates. The lower band is a nonspecific band (arrowhead). (c) RAW264 cells expressing GFP-Rab35 were incubated with or without (0 min) zymosan for various times at 37°C. Cell lysates were prepared and incubated with GST-ANKR. The proteins associated with GST-ANKR were pulled down using glutathione-sepharose beads and analyzed by western blotting (top panel). The middle panel shows aliquots of total cell lysates. The density of the protein bands was measured. The relative protein band intensity of GTP-Rab35 was normalized to total Rab35 (GTP-bound plus GDP-bound forms).

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