GST-PAKcrib is a p21rac activation-specific probe. (A) Recombinant p21rac (0.2 μg) was generated and loaded in vitro with either GDP or the nonhydrolyzable GTP analogue, GppNHp, as described in Materials and Methods. p21rac was isolated using GST-PAKcrib and subsequently detected by Western blot analysis. (Top) GST pull-down of GDP-p21rac (lane 1) or GTP-p21rac (lane 2). (Middle) Coomassie staining of the same blot showing equal GSP-PAKcrib protein in both lanes. (Bottom) A fraction (1/40) of the sample used for the assay was probed with anti-rac antibody to show equal amounts of p21rac were present in both lanes. (B) BW5147 cells (10⁷) stably retrovirally transduced with either control virus or epitope-tagged p21rac(V12)40 were lysed and active p21rac was isolated using GST-PAKcrib as described in Materials and Methods. p21rac was detected by Western blot analysis. (Top) GST pull-down of p21rac from control (lane 1) or p21rac(V12) transfected cells (lane 2). p21rac was detected using anti-rac antibody. (Bottom) A fraction of the lysate used for the pull-down assay was probed with anti-p21rac. (C) (Top) GST pull-down of p21rac from BW5147 cells stably retrovirally transduced with control virus (lane 1), p21rac(V12) (lane 2), p21rho(V14) (lane 3), or p21ras(V12) (lane 4).40 p21rac was detected using an antibody directed against the epitope-tag. (Middle) A fraction of the lysate used for the pull-down was probed with antibody directed against the epitope-tag. (Bottom) The same blot was subsequently probed with anti-rac to demonstrate the specificity of the anti-rac antibody.