Figure 3

mSirt6 interacts with 9-1-1 and hMYH^Q324H does not interact with hHus1, but retains interaction with hAPE1 and mSirt6. a Immobilized GST, GST-tagged hRad9, hHus1, and hRad1 (shown in Additional file 1: Figure S2b) were used to pull-down FLAG-mSirt6. The procedures are similar to those described in Figure 1g. Lane 1 contains 10% of input mSirt6 protein. b Interaction between hSIRT6 and hRad9 is enhanced following H₂O₂ treatment. Rad9 was co-immunoprecipitated by SIRT6 antibody from extracts prepared from untreated (lanes 4) and H₂O₂ treated (lane 6) HeLa cells. Western blots were detected by hRad9 or hSIRT6 antibody. Lanes 1 and 2 contain 10% of input cell extracts (IN). Lanes 3 and 5 are negative control in which the immunoprecipitation was performed with IgG. c Immobilized GST, GST-hMYH(1–350), and GST-hMYH(1–350)^Q324H (shown in Additional file 1: Figure S2C) were used to precipitate hAPE1, mSirt6, and hHus1. Lanes 1 in upper, middle, and lower panels contain 10% of input hAPE1, 20% of input mSirt6, and 10% of input hHus1, respectively. Western blotting was performed with hAPE1, FLAG, or His antibodies. d Quantitative analyses of bound proteins on GST-tagged MYH constructs from three experiments. The Western blots were quantified by the ImageQuant Software (GE Healthcare). Relative interaction was calculated using input in lane 1 as standard. Open bars input references, stripped bars bound proteins on GST-tagged wild-type MYH beads, filled bars bound proteins on GST-tagged mutant MYH beads.