Figure 4.

SIRT6 is a negative regulator of IGF signaling. (a) Representative western blots showing increased expression and phosphorylation of IGF signaling–related proteins in SIRT6 knockout hearts compared to WT control hearts. The expression of p38 and tropomyosin (tropo.) were not changed in these hearts and were therefore used as loading controls. n = 6 mice per group. The antibodies used for detecting ERK and GSK3 recognized the ERK1/2 and GSK3α/β isoforms, respectively. The ‘p-’ prefix indicates the phosphorylated form. (b) Representative western blots showing increased expression of transcription and translation factors in SIRT6 knockout hearts. n = 6 mice per group. (c) Representative western blots showing expression of IGF1R, p-IGF1R and SIRT1 in heart lysates of WT, SIRT1 knockout and SIRT6 knockout mice. n = 3 mice per group. (d) Real-time PCR analysis of IGF signaling–related genes in heart samples of WT and SIRT6 knockout mice. n = 6 mice per group. (e) ChIP analysis to detect SIRT6 binding to the promoters of the indicated genes performed with a SIRT6-specific antibody or IgG control antibody in WT or SIRT6 knockdown (Sirt6KD) cardiomyocytes. The occupancy of SIRT6 to promoters is shown relative to background signals with IgG control antibody. n = 4 independent experiments. Data are presented as the mean ± s.d. Student’s t test was applied to calculate the P value. *P < 0.001.