Figure 3

pIN-KDEL is targeted to the endoplasmic reticulum. HEK293 cells transiently transfected with pIN-G (panels A-D), pIN-MDEL (panels E-H) and pIN-KDEL (panels I-L, P) were fixed at 48 h, permeabilized and treated with antibodies (See Materials and Methods) to the following organelle markers: Sec61β (Endoplasmic Reticulum)(panels A, E, I, M, P); GM130 (Golgi apparatus)(panels B, F, J); EEA1 (Early endosomes)(panels C, G, K); LAMP-1 (Lysosomes) (D, H, L), followed by the appropriate Cy-3 conjugated secondary antibody. Green denotes EGFP fluorescence while red corresponds to the organelle marker. Areas of red/green overlap are shown in yellow. Blue indicates DAPI-stained nuclei. Scale Bar: 15 μM. Cells expressing pIN-KDEL (panels I-L, P) or a commercially available reporter (panel M), show strong overlapping fluorescence with the ER marker Sec61β (panels A, E, I, M). Extensive overlap with the ER is also seen with pIN-KDEL transfected COS-7 cells (panel P). In contrast, pIN-MDEL (panels E-H) and pIN-G (panels A-D) are found throughout the anterograde or entire secretory pathway, respectively. Fluorescence is present throughout the cytoplasm of cells transfected with a plasmid encoding EGFP (panel N) and is absent in cells transfected with an insert-free plasmid (panel O). Scale bar 15 μm.