Fig. 3. More than one binding site is required for localisation of PDE4A1 in the intact cell. (A) Cos1 cells were transfected with engineered constructs formed from either substitution of helix-1 with helix-2 (H1-H1-GFP, green), or by substitution of helix-2 with helix-1 (H2-H2-GFP, green). Cells were stained with BODIPY ceramide (red) or treated with Mitotracker (red) and visualised. (B) Percentage (± s.e.m.) co-localisation of H2-H2-GFP with markers of the Golgi apparatus (BODIPY ceramide), ER (ER tracker) or mitochondria (Mitotracker) (n=25 cells). (C) Cos1 cells were transfected with a construct that encodes (1-25)-4A1-GFP with deletion of the mobile hinge region [8-13 (1-25)-4A1-GFP, green] and co-stained with BODIPY ceramide (red) or treated with Mitotracker (red) and visualised. Merged images are shown and co-localisation is indicated in yellow. Bars, 10 µm.