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. 2011 Dec 6;6(12):e28587. doi: 10.1371/journal.pone.0028587

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Chang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Left panel: MEF cells were transfected with Rac1 mutants including EGFP-Rac1-61L (lane 1 EGFP-Rac1-17N (lane 2), EGFP-Rac1-Y64D (lane 3), or EGFP-Rac1-Y64F (lane 4), or with EGFP (lane 5), or EGFP-Rac1-WT (lane 6). Coomassie blue staining of the membrane (bottom row, left) verified the addition of an equal amount of PBD-GST. Western blotting was done using both anti-Rac1 and anti-GFP antibodies. Right panel: 15 µg of total protein lysates were also analyzed by SDS-PAGE and immunoblotted to verify equal loading and the expression of the transfected EGFP-Rac1 constructs. (B) Four sets of Rac1 activity assay results were used for densitometry and statistical analysis of the difference in activity between Rac1-WT and Rac1-Y64F. Western blots were scanned and Rac1 activity was normalized to the total cellular Rac1. Data shown are mean values±SEM. * denotes p < 0.02. (C) Protein lysates were harvested from MEF (lane 1) or MEF that were transfected with EGFP-Rac1WT (lane 2), EGFP-Rac1-61L (lane 3), EGFP-Rac1-61L/64F (lane 4), or EGFP-Rac1-61L/64D (lane 5). PBD-GST pull down assays were performed with 800 µg of total protein to determine Rac1 activity. The membrane was immunoblotted with anti-Rac1 to demonstrate the level of Rac1 activity, and was then stained with Coomassie blue to ascertain the relative amounts of PBD-GST substrate added to each sample. Expression efficiency of the wild type and mutant EGFP-Rac1 constructs was assayed using 15 µg of total protein from whole cell lysates and the Rac1 activity was normalized to the respective protein level of each sample. (D) Five sets of experimental data were analyzed to examine the differences between the Rac1 activity in lysates from MEF expressing EGFP-RacWT, EGFP-Rac1-61L, EGFP-Rac1-61L/64F, and EGFP-Rac1-61L/64D. ECL blots were scanned and analyzed using Image J software. The intensity of each of the active Rac1 bands pulled down by PBD was normalized to the total cellular Rac protein band and calculated by a software-based algorithm. The Rac1 activity in lysates from cells expressing EGFP-Rac1-61L was assigned a relative value of 100%. ## indicated a significant decrease of Rac1 activity in 61L/64D compared with 61L (p < 0.01). * indicated a significant increase in Rac1 activity in 61L, 61L/64F and 61L/64D compared with Rac1 wild-type (p > 0.001).