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Fig. 2 LC8 facilitates Pak1 nuclear import.
(A) MCF-7 cells transiently transfected with either wild-type (WT), kinase active (T423E) GFP-Pak1, Pak1-LC8mut or Pak1-NLSmut mRNA. Scalebar shown is 10 microns. Mutations of either the NLS or the LC8 binding sequence in WT-Pak1 or T423E-Pak1 markedly reduced EGF-dependent nuclear import and stained with DAPI to visualize nuclei. (B) Quantification of nuclear accumulation of MCF-7 cells harboring either Pak1 or Pak1 mutants. Each bar represents percentage of cells with nuclear localized GFP (50 cells per experiment, done in triplicate). (C) The fraction of GFP located in cytoplasmic (C) and nuclear (N) fractions of MCF-7 cells after stimulation with EGF. Potential cross contamination of nuclear and cytoplasmic fractions was assessed by immunoblot analysis of Laminin A&C and Vinculin, respectively. (D) Nuclear import of T423E-Pak1 mutants after EGF stimulation. Nuclear percentages were calculated as in B. (E) Western Blot analysis of cytoplasmic and nuclear fractions of MCF-7 cells expressing T423E-Pak1 mutants after stimulation with EGF using an anti-GFP antibody.