Genetic interactions in the β-adrenoceptor/G-protein signal transduction pathway and survival after coronary artery bypass grafting: a pilot study  U.H. Frey, E. Kottenberg, M. Kamler, K. Leineweber, I. Manthey, G. Heusch, W. Siffert, J. Peters  British Journal of Anaesthesia  Volume 107, Issue 6, Pages 869-878 (December 2011) DOI: 10.1093/bja/aer302 Copyright © 2011 The Author(s) Terms and Conditions

Fig 1 Reporter assays with alleles from major haplotypes of Caucasians. HEK293 cells were transfected with the respective plasmids, cultivated in medium containing 10% fetal calf serum, and reporter activity was quantified by alkaline phosphatase (SEAP) assays after 24 h. Alleles with their respective positions are shown in the middle. (a) Comparison of constructs 1 (intron 1; 1207–2481) and 2 (intron 1; 1974–2481). (b) Site-directed mutagenesis of haplotype *1 and *2 constructs results in all possible combinations at positions 2291 and 2445. Data are means (sem) from at least three independent experiments each performed in duplicate. *P<0.05, ***P<0.001 Wilcoxon signed-rank test. British Journal of Anaesthesia 2011 107, 869-878DOI: (10.1093/bja/aer302) Copyright © 2011 The Author(s) Terms and Conditions

Fig 2 GNAS allele-specific transcription factor binding and western blot analysis. (a) HeLa and HEK293 nuclear extracts were incubated with equal amounts of probes with different alleles in the absence or presence of a molar excess of unlabelled probe. Representative blot from two independent experiments with similar results. 2291T and 2291C oligos were used for competition gel shift experiments. The 2291T probe evoked one stronger band (complex A) and one additional band (complex B) in contrast to the 2291C probe. Specificity was proven by competition of 100-fold molar excess of the unlabelled probe. The faster migrating complex C shows no specificity as seen from non-competition with the unlabelled probe. (b) 2445G and 2445C oligos were used for competition gel shift experiments. The 2445C probe produced one additional band (complex A) in contrast to the 2445G probe. Specificity was proven by competition of 100-fold molar excess of the unlabelled probe. Again, the faster migrating complex B shows no specificity as seen from non-competition with the unlabelled probe. (c) Erythrocyte membranes were prepared from 31 healthy blood donors with different GNAS diplotypes. Forty micrograms of protein were used for SDS–PAGE and equal total protein loading was verified by Ponceau S-staining of the blot. Representative blots probed with a Gαs antibody. The 45 kDa band represents the short form of Gαs. Note the density differences between different GNAS diplotypes. (d) Relative quantification of Gαs expression in erythrocyte membranes by densitometry of western blots. Comparisons were done using linear anova. British Journal of Anaesthesia 2011 107, 869-878DOI: (10.1093/bja/aer302) Copyright © 2011 The Author(s) Terms and Conditions

Fig 3 Cardiac-specific 1 yr survival of CABG patients. The Kaplan–Meier curves compare the time from CABG surgery to cardiac-specific death or loss to follow-up. (a) GNAS G(-1211)A SNP, (b) GNAS C2291T SNP, and (c) GNAS diplotypes. P-values refer to χ2 statistics for 1 yr cardiac-related survival. British Journal of Anaesthesia 2011 107, 869-878DOI: (10.1093/bja/aer302) Copyright © 2011 The Author(s) Terms and Conditions

Fig 4 Cardiac-specific 1 yr survival of CABG patients. The Kaplan–Meier curves compare the time from CABG surgery to cardiac-specific death or loss to follow-up. (a) ADRB2 Gly16Arg, (b) ADRB2 Glu27Gln, and (c) risk alleles derived from GNAS diplotypes and ADRB2 Gly16Arg genotypes. P-values refer to χ2 statistics for 1 yr cardiac-related survival. British Journal of Anaesthesia 2011 107, 869-878DOI: (10.1093/bja/aer302) Copyright © 2011 The Author(s) Terms and Conditions