Fig. 6. Subcellular localization of a tagged AgARH-like protein. HeLa cells, transiently transfected with the GFP-AgARH-like protein, were fixed and stained with mAb AP.6 directed against the subunit of the AP-2. GFP (green, A,D,F,I) and AP-2 (red, B,D',G,J) signals were collected sequentially by confocal microscopy and representative single optical sections are shown. Scale bar: 20 µm. An enlarged region illustrating the degree of colocalization (D"; arrows) of GFP-AgARH-like fusion (D) with AP-2 (D') is shown, as well as an example of large intracellular aggregates of the fusion protein below the nucleus (F) also containing AP-2 (G,H; arrows). A region where several adjacent cells mechanically detached from the glass surface leaving only adherent ventral plasma membrane (E) shows a high degree of colocalization of the GFP-AgARH-like protein (yellow) compared with the non-transfected cells (red). The generally diffuse intracellular distribution of the doubly mutated GFP-AgARH-like (LIDAAA/W268A) mutant is shown (panels I-K).