Fig. 6

Effect of expression of COPB1 mutation on in vitro localisation and stability of β-COP protein. a Western blot image of protein extracts from HEK293 cells transfected with wild-type, and c.1651T>G (COPB1 tagged with c-myc, immunoblotted with c-myc antibody to detect COPB1, and with beta-actin antibody as loading control). c.1651T>G shows a slightly reduced normalised level. b Immunofluorescence images of hTERT-RPE1 cells nucleofected with wild-type COPB1 (top row), and COPB1 c.1651T>G p. p.Phe551Val (lower row) tagged with c-myc. Cells were stained with Golgi stain Bodipy-TR-ceramide (red), nuclear stain DAPI (blue) and immunostained with anti-myc (green). Mutant expression shows more diffuse β-COP staining throughout the Golgi and outside the Golgi in the wider cytoplasm. Scale bar = 5 μm. c Immunofluorescence images of hTERT-RPE1 cells nucleofected with wild-type COPB1 (top row), and COPB1 c.1651T>G p. p.Phe551Val (lower row) tagged with c-myc. Cells were stained with nuclear stain DAPI (blue) and immunostained with an antibody to resident Golgi protein giantin (green) and anti-β-COP (red). This clarified that wild-type β-COP tended to localise to the edges of the Golgi stacks, whereas mutant β-COP was localised throughout the Golgi. Scale bar = 10 μm