Research Paper Volume 14, Issue 20 pp 8179—8204

Figure 3. SIX2/SIRT1/AKT/GSK3β network is altered in UdRPCs derived from aged donors. Relative protein expression normalized to ß-ACTIN for SIRT1, AKT and GSK3ß and relative protein phosphorylation for AKT, GSK3β and pH2A.X was detected by Western blot (A). mRNA expression of SIRT1 was determined by quantitative real time PCR (B). Detailed analyses by bisulfite sequencing of CpG island methylation patterns within 5′ regulatory region of SIRT1 gene in young and aged UdRPCs (C). SIX2 binding within the SIRT1 gene was confirmed by Immunoprecipitation followed by PCR analysis (D). The grey dashed line indicates that gel picture has been merged (for the original gel picture see Supplementary Figure 6).