Immunoblot identification of E- and N-cadherin in the gel-electrophoretically separated polypeptides and protein complexes of whole-cell lysates, sucrose density centrifugation, and IP fractions of mammalian liver tissue and cultured hepatocellular tumor cells. (A) On SDS-PAGE separation (8% gel) of total proteins, E-cadherin (E-cad) and N-cadherin (N-cad) are detected as distinct bands of ∼135 kD in the following human cells and tissues: hepatocellular carcinoma cells of the line primary liver carcinoma (PLC; lane 2), human hepatoblastoma-derived cells of the line HepG2 (lane 3), human hepatocellular carcinoma cells of the lines Hep3B (lane 4) and HuH7 (lane 5); primary cultures of human hepatocytes (phH; lane 6), human liver tissue (hLiver; lane 7), and a solid human hepatocellular carcinoma (HCC; lane 8). Different detergent-soluble fractions from bovine liver (bLiver; RIPA or Triton buffers; lanes 9 and 10, respectively) have also been analyzed. E-cadherin is not detected in cells of human hepatic stellate cells of line LI-90 (lane 1) and of the human glioma cell line hG-U333 (lane 11) analyzed in parallel but is a major component in cultured cells of the human colon carcinoma line CaCo-2 (lane 12). N-cadherin is detected in all hepatocyte and hepatocyte-derived cells as well as in human hepatic stellate cells of line LI-90 and in the positive controls of human glioma cells (hG-U333) but not in CaCo-2 cells. (B) Sucrose density centrifugation (5–30%) of particles obtained by solubilization of total lysates of PLC cells, followed by fractionation (fraction numbers are given), SDS-PAGE of the fractions obtained, and immunoblotting with antibodies against E-cadherin (top) and N-cadherin (bottom). Note that for both cadherins, the maximum peak is near that of catalase (the positions of proteins analyzed in parallel are given at the top margin: BSA [B] with 4.3 S, catalase [C] with 11.5 S, and thyroglobulin [T] with 16.5 S). (C) Immunoblots of proteins contained in the peak fractions 5–8 of the gradient shown in A and obtained by IP with antibodies to E-cadherin (lane E) or N-cadherin (lane N). Apparent molecular masses according to SDS-PAGE mobility are 135 kD (N-cadherin) and 124 kD (E-cadherin). Molecular masses of reference proteins examined in parallel are indicated on the left. White lines indicate that intervening lanes have been spliced out.