Subcellular distribution of ENS-1 in CES and during differentiation.

<p>(<b>A</b>) Immunostaining of ENS-1 (16h4 antibody) and HP1γ (Abcam antibody) in CES (-RA) and CES differentiated with retinoic acid for the indicated period. Nuclei are labeled with Draq5 and are in blue in merging panels. Image acquisition was optimized to observe the distribution of the proteins and does not reflect the real expression level. Scale bar 15 μm. (<b>B</b>) Western blot analysis of ENS-1 in the cytoplasmic (Cy) and in the nuclear (Nu) fractions of undifferentiated CES (-RA) or CES induced to differentiate with retinoic acid for 24h or 48h. Ponceau's red staining serves as a protein loading control between both fractions. In the nucleus soluble and insoluble components were separated and only the precipitating fraction that contains ENS-1 is shown. The volume corresponding to 6×10⁶ cells was loaded for the N fraction and Lamin B1 was used as loading control. In the cytoplasm (Cy) ENS-1 was in the supernatant, 15 μg of proteins were loaded and HSP90 was used as loading control. The vertical line indicates missing lanes between the two presented parts of the same gel. Dotted lines in red indicate the position of three ENS-1 related proteins. (<b>C</b>) Separation of soluble and insoluble components from the cytoplasm. The cytoplasmic fraction separated from the nucleus was subjected to extended centrifugation. The pellet (Cy p) and the supernatant (Cy s) were analyzed for ENS-1 protein and HP1γ. The nuclear fraction corresponding to the whole nuclear proteins is represented (Nu). Protein loading was 10 μg for each fraction. HSP90 identifies the cytoplasmic soluble fraction, Lamin B1 identifies fractions containing membrane proteins and 2MeH3K4 identifies fractions containing chromatin. Results in (B) and (C) were obtained from two independent experiments using distinct fractionation protocols. (D) Summary tables of the ENS-1 forms found in the cytoplasm (upper table) and in the nucleus (lower table) from CES cells (-RA) and from CES induced to differentiate with retinoic for 24 h or 48 h.