Figure 5. Western blot analysis of the protein ENS-1.
(A) Proteins (15 μg) of whole cell lysates from murine (MES) and chicken (CES) cells were analyzed by western blot using the anti-ENS-1 3807 antibody. Protein loading was equivalent in both conditions as illustrated by the Ponceau's red staining of the blot. (B) Proteins lysates (20 μg) from CES cells transfected with an HA-tagged ENS-1 protein (lanes 4,5,6) or with an empty vector (lanes 1,2,3) were compared with untransfected cells (WT, lane 7). Triplicates were from three independent transfection experiments. The HA-tagged protein was detected by the 3807 antibody and by the anti-HA antibody at a molecular weight of 60 kDa. The anti-ENS1 antibody also detected the endogenous protein at 55 kDa. Both proteins differed in size by 3 kDa corresponding to the 33 additional amino acids added at the C-terminal part of ENS-1 in the transgenic protein in addition to the two HA tags (2.4 kDa). (C) Western blot analysis of ENS-1 and HP1γ (Chemicon antibody) in whole cell lysates (12 μg) from undifferentiated CES (-RA) or CES induced to differentiate with retinoic acid (RA, 10−6M) for 4 to 48 h as indicated. HSP90 was used as protein loading control.