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The pellet was lysed, and soluble Rac1-GTP was recovered using PAK1-functionalized beads, then labeled with anti-Rac1 primary and fluorescent secondary antibodies before being analyzed using a flow cytometer

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Similarly, replacing the 2-chloroacetamide with a 3-chloroacetamide derivative such as in cluster-4 also proved unfavourable and resulted in loss of potency