Fig. 2. Identification of histone H3.3A as a Pak1 substrate. (A) G₂–M expression library screening by Pak1. (B) mRNA expression of H3.3A in breast cancer cell lines and murine tissues. (C) Direct interaction of Pak1 with histone H3 in a GST pull-down assay. In vitro-translated H3 bound to GST–Pak1 (left panel), and in vitro-translated Pak1 bound to GST–H3 (right panel). (D) Non-interaction of H3 with Pak 2 and Pak3. cDNAs were translated in vitro, incubated with GST–H3 and analyzed by a GST pull-down assay. (E) Interaction of Pak1 domains with histone H3. Pak1 GST-fusion proteins containing full-length Pak1 amino acids (aa 1–546), the kinase domain (aa 270–546), the N-terminal domain (aa 1–132), the Nck-binding domain (aa 1–75), the Cdc42/Rac-interacting domain (aa 52–132) and the autoinhibitory domain (aa 75–149) were all incubated with in vitro-translated H3, and binding was analyzed by a GST pull-down assay. (F) Pak1 specifically interacts with H3 but not H4 in TNT pull-down assays. GST pull-down assay using ³⁵S-labeled Pak1 and GST fusion of H3 or H4. (G) Pak1 interacts with H3 tail. GST pull-down assay using ³⁵S-labeled Pak1 and GST fusions of H3 1–57 or H3 1–29.