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The activities of many transcription factors are controlled by intracellular signal transduction pathways that respond to changes in the extracellular environment. The resulting changes in gene expression lead to appropriate physiological responses. Elucidating the signaling pathways that target transcription factors is an important goal as they represent potential targets for therapeutic intervention in many diseases. A number of tools and techniques have been developed that can be used for studying signaling specificity toward transcription factors. This article describes how (1) mutant alleles of signaling enzymes and (2) protein/peptide inhibitors can be used in conjunction with phosphorylation-specific antibodies and reporter gene assays to examine the targeting of transcription factors by signaling pathways in vivo.