Figure_6.tif (1.46 MB)
Klf3H275R mRNA, protein, and impairment in DNA binding.
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posted on 2013-07-11, 03:53 authored by Lois Kelsey, Ann M. Flenniken, Dawei Qu, Alister P. W. Funnell, Richard Pearson, Yu-Qing Zhou, Irina Voronina, Zorana Berberovic, Geoffrey Wood, Susan Newbigging, Edward S. Weiss, Michael Wong, Ivan Quach, S. Y. Sandy Yeh, Ashish R. Deshwar, Ian C. Scott, Colin McKerlie, Mark Henkelman, Peter Backx, Jeremy Simpson, Lucy Osborne, Janet Rossant, Merlin Crossley, Benoit Bruneau, S. Lee Adamson(A) Similar levels of Klf3 mRNA by qRT-PCR (P>0.05) and (B) KLF3 protein by Western blot (WB) in whole H275R homozygotes (Homo) and heterozygotes (Het) versus wild-type (WT) embryos at E12.5. (C) Bacterial expression of GST-zinc finger 1–3 of normal and mutant protein showing that H275R impaired DNA binding to CCACACCCT (canonical β-globin promoter) in electrophoretic mobility shift assay (coomassie blue-stained SDS PAGE gel (below)). (D) Dose-response showing H275R also impaired binding to CCACACCT in electrophoretic mobility shift assays when full length Klf3 was expressed in COS cells (western blot with αKlf3 antibody below). Protein identity was validated by eliminating binding with a KLF3 antibody (αKlf3).
