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. 2001 Feb;12(2):279–295. doi: 10.1091/mbc.12.2.279

Figure 6.

Figure 6

Loss of Na,K-ATPase β-subunit and E-cadherin expression in a reverted clone of MSV-MDCK cells expressing Na,K-ATPase β-subunit and E-cadherin, and characterization of MSV-MDCK cells expressing E-cadherin and Na,K-ATPase α-subunit. (A) Total cell lysates (50 μg) were separated on a 10% SDS-PAGE and immunoblotted for Na,K-ATPase β-subunit as described in MATERIALS AND METHODS. Reversion of MSV-Cad-NaKβ-cl 1 cells (MSV-Cad-NaKβ rev) results in a drastic reduction of β-subunit expression. (B) Total cell lysates (100 μg) were separated on a 10% SDS-PAGE and immunoblotted for E-cadherin. MSV-Cad-NaKβ rev cells show also a dramatic reduction of E-cadherin protein levels. (C) Loss of E-cadherin and β-subunit expression results in the reversion to a fibroblastic phenotype. Bar, 50 μm. (D) Transmission electron microscopy reveals that MSV-Cad-NaKβ rev cells form neither tight junctions nor desmosomes (arrows). Bar, 0.5 μm. (E) Immunoblot analysis: total cell lysates (100 μg) of MSV-Cad-NaKα cells and MDCKwt cells were separated on a 10% SDS-PAGE and immunoblotted for Na,K-ATPase α-subunit as described in MATERIALS AND METHODS. (F) Cell surface biotinylation: Cells were biotinylated and immunoblotted for Na,K-ATPase α-subunit. (G and H) Immunofluorescence staining of ZO-1. In MSV-Cad-NaKα-cl 1 (G) and in MSV-Cad-NaKα-cl 2 (H) cells, ZO-1 is localized on the plasma membrane but does not show a continuous ring-like staining pattern. Bar, 10 μm.