American Society of Hematology

Fig. 2.

$Fig. 2. The GST-PAKcrib assay can be used to measure p21rac activation in human neutrophils. (A) Human neutrophils were isolated as described and stimulated with fMLP (1 μmol/L) for 10 seconds as indicated. Cells were lysed and GST pull-down was performed using either GST or GST-PAKcrib as described in Materials and Methods. (Top) The isolated p21rac was detected by Western blot analysis using anti-rac antibody. (Middle) A Coomassie staining of the blot. The arrowheads indicate the GST and GST-PAKcrib proteins. (Bottom) A fraction of the lysate used for the pull-down was probed with anti-Rac. (B) Neutrophils were isolated as described and stimulated with fMLP (1 μmol/L) for the indicated time. Cells were lysed and active p21rac was isolated using GST-PAKcrib as described in Materials and Methods. (Top) The GST-PAKcrib–associated p21rac was detected by Western blot analysis using anti-Rac antibody. (Middle) A fraction of the lysate used for the pull-down was probed with anti-Rac. (Bottom) Coomassie staining of the blot, the arrowhead indicates the GST-PAKcrib protein.$

The GST-PAKcrib assay can be used to measure p21rac activation in human neutrophils. (A) Human neutrophils were isolated as described and stimulated with fMLP (1 μmol/L) for 10 seconds as indicated. Cells were lysed and GST pull-down was performed using either GST or GST-PAKcrib as described in Materials and Methods. (Top) The isolated p21rac was detected by Western blot analysis using anti-rac antibody. (Middle) A Coomassie staining of the blot. The arrowheads indicate the GST and GST-PAKcrib proteins. (Bottom) A fraction of the lysate used for the pull-down was probed with anti-Rac. (B) Neutrophils were isolated as described and stimulated with fMLP (1 μmol/L) for the indicated time. Cells were lysed and active p21rac was isolated using GST-PAKcrib as described in Materials and Methods. (Top) The GST-PAKcrib–associated p21rac was detected by Western blot analysis using anti-Rac antibody. (Middle) A fraction of the lysate used for the pull-down was probed with anti-Rac. (Bottom) Coomassie staining of the blot, the arrowhead indicates the GST-PAKcrib protein.

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