FIGURE 2.

Cultivation temperature does not affect cell surface targeting of Eag channels. A, cell surface proteins were biotinylated using a membrane-impermeable reagent and isolated with streptavidin beads. Channel immunoreactivity in the streptavidin precipitates (1st panel) and in the lysates (3rd panel) was detected with an anti-Eag antibody that recognizes the EagCT. The biotinylation reagent did not label an intracellular protein, GAPDH (2nd and 4th panels), confirming its specificity for cell surface proteins. B, temperature does not affect the proportion of Eag or Eag-NTmEag trafficked to the plasma membrane. Both Eag and Eag-NTmEag are targeted to the plasma membrane in similar proportion to cells cultivated at 37 °C (white bars) and 26 °C (black bars). Data are expressed as the ratio of arbitrary densitometry units from streptavidin precipitates and cell lysates as determined by densitometric analyses of immunoblots. C, confocal images of tsA 201 cells that were either untransfected or transfected with eag-EX-myc cDNA. Anti-Myc antibodies were used to label surface (top panels) and total Eag protein (bottom panels). Representative images from one of six independent experiments are shown.