Figure 3

Visualization of N- and C-termini in S2 cells shows a difference in the subcellular localization of Ths and Pyr C-terminus. S2 cells were transfected with the indicated pUASt.HA-Pyr-Myc, pUASt.Pyr-GFP, pUASt-Ths-GFP or pUASt.HA-Ths-Myc constructs (see methods), and immunofluorescence was conducted using anti-HA, anti-Myc, or anti-GFP antibodies (all "red"). HA-Pyr-Myc was stained with anti-HA to see the N-terminus (A,D) where predominant ER staining was seen inside the cell (A) while only membrane staining was seen under non-permeabilizing conditions (D). The C-terminus of Pyr was visualized with anti-Myc for HA-Pyr-Myc (B,E) or anti-GFP for Pyr-GFP (C,F). The C-terminus of Pyr inside the cell was localized to small, non-nuclear vesicles, which may be endosomal in character (B,C). No Pyr C-terminus was visualized outside of the cell (E,F). Anti-HA was used to visualize the HA-Ths-Myc N-terminus under permeabilizing (G) and non-permeabilizing conditions (J), revealing ER staining around the nucleus inside the cell (G) and proteins attached to the cell membrane (J). Anti-Myc (H,K) and anti-GFP (I,L) were both used to visualize the Ths C-terminus of either HA-Ths-Myc or Ths-GFP. Again, ER staining was seen inside the cell (H,I) and membrane staining was observed under non-permeabilized conditions (K,L).