Figure 2. Association of the C-terminal... | Rockefeller University Press

Figure 2.

$Figure 2. Association of the C-terminal part of RasGEF Q with F-actin. (A) The cortical localization of GFP-GEF is sensitive to LatA treatment. Cells expressing GFP-GEF (green) were incubated with LatA for 40 min, fixed with picric acid/formaldehyde, and stained for actin using TRITC-labeled phalloidin (red). Images were taken using a confocal microscope. (B) GST-GEF binds to F-actin in an in vitro cosedimentation assay using D. discoideum actin. Pellet (P) and supernatant (S) were separated by high-speed centrifugation, and proteins in the fractions were resolved by SDS-PAGE and stained with Coomassie brilliant blue. (C) Cytoskeletal fractions were prepared from AX2 cells expressing GFP-GEF using a buffer containing 2% saponin. Total cell lysates (L), pellet (P), and supernatant (S) fractions were resolved on SDS-PAGE and amounts were analyzed by Western blotting using a GFP monoclonal antibody. Coomassie-stained gel showing the actin band is used as control. (D) Effect of drugs affecting the cytoskeleton on localization of GFP-RasGEF Q173. Cells expressing GFP-RasGEF Q173 (green) were treated with DMSO (control; top row), 10 μM LatA (second row), 20 μM cytochalasin D (third row), or 100 μM Blebbistatin (fourth row) for 40 min. Cells were fixed with cold methanol. Actin (red) was recognized by mAb Act1–7 followed by cy3-labeled anti–mouse secondary antibody. Carats point to areas of colocalization. Bars, 10 μm.$

Association of the C-terminal part of RasGEF Q with F-actin. (A) The cortical localization of GFP-GEF is sensitive to LatA treatment. Cells expressing GFP-GEF (green) were incubated with LatA for 40 min, fixed with picric acid/formaldehyde, and stained for actin using TRITC-labeled phalloidin (red). Images were taken using a confocal microscope. (B) GST-GEF binds to F-actin in an in vitro cosedimentation assay using D. discoideum actin. Pellet (P) and supernatant (S) were separated by high-speed centrifugation, and proteins in the fractions were resolved by SDS-PAGE and stained with Coomassie brilliant blue. (C) Cytoskeletal fractions were prepared from AX2 cells expressing GFP-GEF using a buffer containing 2% saponin. Total cell lysates (L), pellet (P), and supernatant (S) fractions were resolved on SDS-PAGE and amounts were analyzed by Western blotting using a GFP monoclonal antibody. Coomassie-stained gel showing the actin band is used as control. (D) Effect of drugs affecting the cytoskeleton on localization of GFP-RasGEF Q¹⁷³. Cells expressing GFP-RasGEF Q¹⁷³ (green) were treated with DMSO (control; top row), 10 μM LatA (second row), 20 μM cytochalasin D (third row), or 100 μM Blebbistatin (fourth row) for 40 min. Cells were fixed with cold methanol. Actin (red) was recognized by mAb Act1–7 followed by cy3-labeled anti–mouse secondary antibody. Carats point to areas of colocalization. Bars, 10 μm.