Establishment of cell lines stably expressing the ccp1 protein. Cell lines stably expressing ccp1/eGFP fusion proteins were generated using retroviral approach. (A) The expression of ccp1/eGFP fusion protein was analyzed in MEF cell lines using polyclonal sera raised against ccp1 (left panel) and GFP antibody (right panel). The ccp1/eGFP fusion protein was detected as a single band at the expected molecular weight of 55 kDa. (B) The expression of ccp1/eGFP fusion protein was also analyzed in neuroblastoma, SK-N-SH cell lines using GFP antibody. 7.5% SDS-PAGE. (C) MEF (a-d, i-j) and SK-S-NH (e-h) were cultured in the presence of serum. (a-b, e-f) Cells were analyzed using anti-GFP antibody and confocal microscopy. (a) Mostly nuclear and weak cytoplasmic localization was diffusely observed in the eGFP protein stably expressed in MEF. (b) The ccp1/eGFP fusion protein stably expressed in MEF was observed in the cytoplasm in punctate spots. (c) Control MEF presented a typical fibroblast flat shape. (d) MEF expressing ccp1/eGFP fusion protein appeared smaller and shaped spindle-like. (e-h) Similar pattern was observed in SK-N-SH cells. (i-j) Filamentous actin was visualized with Teas Red-conjugated phalloidin (red) in MEF control and MEF-ccp1/eGFP. Nuclei were stained using DAPI (blu). Scale bar in a-b, e-f, i-j = 25 μm (line) and in c-d, g-h = 200 μm (dotted line).