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Fig. 11. Accumulation of ErbB2 in Arf-Q67L-positive endosomes occurs only without GA. (A-F) SK-BR-3 cells were transfected with Arf6-Q67L alone (A,D) or in combination with either GFP-Rab5 (B,E) or GFP-Rab7 (C,F). Fl-anti-ErbB2 (A,D) or unlabeled anti-ErbB2 (B,C,E,F) antibodies were bound for 1 hour before cells were warmed for 2 hours with (A-C) or without (D-F) GA. Internalized anti-ErbB2 was detected in fixed and permeabilized cells by Fl-anti-ErbB2 fluorescence (A,D) or with Texas-Red goat-anti-mouse antibodies (B,C,E,F). Although Arf6-Q67L was not visualized in B,C,E or F, vacuoles characteristic of Arf6-Q67L expression were seen. Scale bar: 10 µm. (G,H) COS-7 cells were transfected with ErbB2 alone or together with Arf6-Q67L as indicated. Cells were incubated with GA for the indicated times and solubilized in gel loading buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. ErbB2 and Arf6-Q67L were detected by immunoblotting as described in the Materials and Methods. (H) A representative Arf6-Q67L blot is shown, demonstrating expression in co-transfected cells (+Arf6-Q67L), but not in cells expressing ErbB2 alone (–Arf6-Q67L).
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