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. 2013 Aug 5;8(8):e70891. doi: 10.1371/journal.pone.0070891

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© 2013 Nin et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A, Serine to alanine substitution at 1450 abrogated Akt-induced N-CoR misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450A or T1925A mutants) in 293T cells transfected with the constitutively active myr-Akt was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions were separated by high-speed centrifugation and N-CoR levels in each fraction were determined with flag antibody (upper panel). The relative solubility/insolubility of β-actin in each fraction was determined as a control (middle panel). The level of total of protein in each fraction was determined by coomassie blue staining (lower panel). B, N-CoR S1450E, the phosphomimetic mutant of N-CoR, displayed signs of misfolding. Relative solubility/insolubility of flag-tagged N-CoR (WT and S1450E) in 293T cells was determined by protein solubility assay. C, S1450E is localized in cytosol. Subcellular distribution of flag-tagged N-CoR (WT, S1450A and S1450E) was determined by confocal microscopy after staining of the cells transfected with each plasmid with flag antibody. D, S1450E can induce ER stress. Level of GRP78/BiP (green) in 293T cells transfected with S1450E (red) was determined by confocal microscopy after staining the cells with flag and GRP78/BiP antibodies.