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. Author manuscript; available in PMC: 2011 Mar 30.
Published in final edited form as: J Biol Chem. 2000 Sep 29;275(39):30740–30745. doi: 10.1074/jbc.M001702200

Fig. 2. Raf1-RBD-GST recognizes GTP-bound Ha-, K-, and N-Ras.

Fig. 2

NIH 3T3 cells were transfected with pCGN-hyg constructs encoding HA-tagged Ha-Ras (C and D), K-Ras (E and F), or N-Ras (G and H) or the HA tag alone (A and B) and then grown for 24 h. Cells were then switched to serum-free medium for 48 h, processed for HA immunocytochemistry (A, C, E, and G) and Ras-GTP analysis (B, D, F, and H), and then examined by fluorescence microscopy. Note that cells transfected with all three HA-tagged Ras species show high levels of Raf1-RBD-GST immunofluorescence, compared with cells transfected with HA tag alone. Untransfected NIH 3T3 cells grown in the absence of serum for 48 h demonstrated only background staining when examined by Raf1-RBD-GST immunocytochemistry (I). However, after treatment for 2 min with 20 ng/ml insulin (J), these cells demonstrated increased, punctate immunofluorescence that was localized in patches, apparently at the cell membrane (see “Results”). These data indicate that Raf1-RBD-GST can detect elevated Ras-GTP in situ.