PMC

Phospho immuno reactivity of GRK2 in D153del affected retina, brain, heart liver and kidney show tissue specific changes in rge chicken protein extracts compared to wt controls. Experiments represent a total of n = 3 independent experiments. Statistical significance is shown as * P<0.05, **P<0.01, ***P<0.001.

Relative degradation of GNB3YFP and GNB3dYFP in COS7 cells at different time points after treatment with cyclohexamide and probed against custom raised GNB3 antibody. The graphs are representative of a total of three independent experiments and relevant readings at each individual time point were normalised to the densitometry units of endogenous GNB3 on the same blot.

COS-7 cells transiently transfected with recombinant constructs expressing GNB3YFP (Fig. 1a–d) and GNB3dYFP (Fig. 1e–h). Cells expressing GNB3 show ER and Golgi, localisation (1i & j). Cells expressing GNB3d mutant protein show predominant localisation to ER (Fig. 1k) but not in Golgi (Fig. 1l). Plasma membrane staining revealed no colocalisation of GNB3dYFP (Fig. 1n) when compared to GNB3YFP (Fig. 1m). These are representative images taken from different fields of view under 40× objective with 1.5× further magnification. Bar = 8 µm.

Immunoreactivity of GNB3 on D153del affected (rge) and normal tissue (wt) protein extracts blotted against Anti-GNB3 followed by quantitative densitometry analyses in the D153del affected pooled tissue samples (n = 8) of the same age. Data presented in all panels are the mean with standard error (bars) of n = 3 independent experiments. Statistical significance is shown as * P<0.05, **P<0.01, ***P<0.001.

Phospho immuno reactivity of ERK2 and AKT in D153del affected retina, brain, heart liver and kidney. Mutant rge chicken protein extracts showed significant changes in relative phosphorylation activity when compared to normal age matched controls that was confirmed by semi quantitative densitometry analyses. Experiments represent a total of n = 3 independent experiment. Statistical significance is shown as * P<0.05, **P<0.01, ***P<0.001.

Immunohistochemistry of GNB3 (green) on kidney sections shows high expression in the epithelial border of the tubules, proximal convoluted tubule (PCT) and glomeruli (GL) of normal kidneys (Fig. 8a), which are less evident in rge sections (Fig. 8b). Postive control staining of COX IV antigen (red) is observed in both rge and wt sections (Fig. 8c & 8d). Figures are representative of different images taken from different fields of view in three individual experiments (n = 3). Magnification 60×.

Glomeruli from mutant kidneys (Fig. 7a) are considerably larger than their wild type counterparts (Fig. 7b); whereas tubules are of approximately the same diameter. Red blood cells in large numbers are seen in the lumen of tubules and where present are associated with flattening injury to the tubular epithelium (Fig. 7c and d). These red cells are not seen in wild type kidneys (Fig. 7b, d, & f). There is a focus of severe tubulo-interstitial inflammation (lower arrow) comprising lymphocytes and macrophages infiltrating tubules in rge that are not seen in wt kidneys. Magnification 66×.

a) Tissue specific regulation of cGMP levels are shown by a significant fold increase in D153del affected retina, but decrease in other D153del tissues compared to unaffected controls. Data are the means and standard error (bars) of n = 3 independent experiments performed in octuplets. Statistical significance is shown as * P<0.05, **P<0.01, ***P<0.001. b) Tissue specific regulation of cAMP levels are, shown by a significant fold increase in D153del affected retina and brain samples and decreased in D153del affected heart, liver and kidney, when compared to levels of normal controls. Data are the mean with standard error (bars) of n = 3 independent experiments performed in octuplets. Statistical significance is shown as * P<0.05, **P<0.01, ***P<0.001.