Figure 2

Identification and determination of the effects of the FKBP5 gene mutation. (a) Sanger sequencing data of codons 160–168 in exon 4 of FKBP5 in a healthy member and a patient, illustrating the presence of an obvious c.163G>C heterozygous mutation in the patient (indicated by the orange arrow). (b) Amino-acid sequence alignment comparing the sequence of FKBP51 in 13 vertebrates. The alignment illustrates the highly conserved nature of the V55 residue in FKBP51. Positions identical to Homo sapiens are highlighted in blue. (c) Three-dimensional structure of the FK1 domain of the wild-type and mutant FKBP51 proteins. The yellow arrow indicates the position of the V55L mutation in which valine is replaced by leucine. (d) Relative luciferase activity of transfected HEK293 cells. There is no significant difference in the luciferase activity of cells transfected with wild-type and mutant FKBP51 (P=0.3624). The data are representative of at least three independent experiments. (e) Western blot showing that both wild-type and mutant FKBP51 are overexpressed in transfected HEK293 cells. (f) A representative western blotting result of Akt phosphorylation in transfected HEK293 cells; the blot shows a higher pAktser473 level in the cells transfected with mutant FKBP51 than in cells transfected with the wild-type protein. (g) Band intensity ratios of phosphorylated AktSer473 to total Akt determined by western blotting. The result shows that the mutant FKBP51 increased Akt phosphorylation in HEK293 cells (P=0.0075). The data are representative of at least three independent experiments. **P<0.01. (h) Phosphorylated GSK-3α detected by western blotting. The blot shows the phosphorylation levels of exogenous GSK-3α phosphorylated by endogenous Akt, which reflects the kinase activity of Akt, in U937 cells expressing wild-type and mutant FKBP51. This result proves that Akt kinase activity in mutant FKBP51-transfected U937 cells is higher than in cells expressing the wild-type protein.