FIGURE 6.

The greater intracellular retention of YFP-β₂ than YFP-β₁ is not associated with its higher level in the plasma membrane. A and B, the comparative Western blot analysis (A) of total cell lysates and basolateral biotinylated proteins (BL membrane) of MDCK cells stably expressing either YFP-β₁ or YFP-β₂ shows that the upper band found in either cell lysate represents the mature plasma membrane fraction (PM), whereas the lower band corresponds to the intracellular fraction ER-resident fraction (ER). Treatment with Endo H resulted in a slight increase in electrophoretic mobility of BLM YFP-β₂, but not of BLM YFP-β₁, and a major increase in electrophoretic mobility of ER YFP-β₁ and ER YFP-β₂. This allows a better separation of PM and ER fractions of YFP-β₁ or YFP-β₂ on SDS-PAGE and their densitometric quantification (B). C, Western blot analysis of proteins isolated by basolateral surface-selective biotinylation show that stable expression of either YFP-β₁ or YFP-β₂ in MDCK cells resulted in a significant decrease in the amount of the endogenous Na,K-ATPase β₁ subunits in the basolateral membranes but did not change the level of the α₁ subunits, as compared with non-transfected cells (NT cells). YFP-β₁ and YFP-β₂ are present in the basolateral membrane at similar levels in the two transfected cell lines. PM, basolateral plasma membrane.