Fig. 4.
Polarized secretion of a soluble protein is not affected by nucleofection. Filter-grown MDCK cells were subjected to Amaxa nucleofection with the indicated siRNA duplexes. The following day, nucleofected and control untransfected cells were plated on filters and protein secretion of a truncated mutant of endolyn (ensol) tagged with GFP was analyzed 4 days later. Cells were radiolabeled with [35S]cys for 30 min and chased for 1.5 h. The apical and basolateral media were collected separately, and the cells were solubilized. Samples were immunoprecipitated using anti-endolyn antibody and analyzed by SDS-PAGE. Representative samples from one experiment are shown and the quantitation of apically secreted ensol in 3 independent experiments performed in triplicate is noted below each condition. A, apical; B, basolateral; C, cell.