Figure 5.

Expression and localization of PLN-WT and PLN-L39stop mutant in HEK-293 cells. (a) Immunoblot analyses of endoplasmic reticulum microsomes (left panel) and insoluble fractions (right panel) obtained at 24 and 48 hours after transfections. Note the absence of PLN-L39stop in the endoplasmic reticulum fraction, although detectable protein levels are found in the insoluble fraction. PLN_p, PLN pentamer; PLN_m, PLN monomer. (b) Quantitation of the number of fluorescent cells on PLN-WT and PLN-L39stop transfected coverslips. Cultures were transfected with equal amounts of plasmid DNA, and 48 hours later the number of fluorescent cells was counted on the entire coverslip. (c) Immunofluorescence of PLN-WT and PLN-L39stop transfected cells analyzed by confocal microscopy 48 hours after transfection. In PLN-WT transfected cells, immunofluorescence is exclusively in the endoplasmic reticulum, whereas a clearly distinct and plasma membrane–associated staining pattern is observed in the PLN-L39stop transfected cells. Scale bar, 30 μm. (d) Fluorescence intensity was quantitated using available Leica software. For these assays, a straight profile line was drawn across the center of the cell and fluorescence amplitude was plotted. Arrows mark the edge of the cell and asterisks mark the ER. In PLN-WT transfected cells (upper panel), immunofluorescence is found within the interior of the cell, whereas in PLN-L39stop transfected cells (lower panel), staining is found enriched at the outer edge of the cell.