Figure 4
Association of SIRT6 with MYH is stabilized by APE1 and Hus1. a–c MYH/SIRT6 complex formation is enhanced by APE1. GST-hMYH(1–350)-WT (a) or GST-hMYH(1–350)V315A (b) (shown in Additional file 1: Figure S2C) were used to pull-down mSirt6 in the presence of hAPE1. Lanes 1 in both (a) and (b) contain 10% of input mSirt6 protein. Lanes 2 in both (a) and (b) are GST alone. Western blots were detected by FLAG antibody. c Quantitation of the relative amount of Sirt6 in the precipitates from three experiments as in a and b. At an 1:1 (lane 4) and 1:10 (lane 5) molar ratio of mSirt6:hAPE1, the amount of mSirt6 pulled down by GST-hMYH-WT increases, but remains the same by GST-hMYHV315A. d–f MYH/APE1 complex formation is enhanced by SIRT6. Reactions are similar to a–c. GST-hMYH(1–350)-WT (d) or GST-hMYH(1–350)V315A (e) (lanes 3–5) were used to pull-down hAPE1 in the presence of mSirt6. Western blots were detected by APE1 antibody. g–i MYH/SIRT6 complex formation is enhanced by Hus1. Reactions are similar to a–c. GST-hMYH(1–350)-WT (G) or GST-hMYH(1–350)Q324H (h) (shown in Additional file 1: Figure S2C) (lanes 3–5) was used to pull-down mSirt6 in the presence of Hus1. Western blots were detected by FLAG antibody. j MYH/SIRT6 complex formation is enhanced by APE1 and Hus1. Reactions are similar to a. GST-hMYH(1–350)-WT was used to pull-down mSirt6 in the combination of APE1 and Hus1. Western blots were detected by FLAG antibody. k Quantitation of the relative amount of Sirt6 in the precipitates from three experiments as in j. The quantitation in c, f, i, and k is similar to that described for Figure 3c.