Figure 2

SMAR1 modulates the deacetylation of Ku70 via HDAC6. (a) Acetylation-specific IP assay in control cells, cells overexpressed for SMAR1 (Ad-SM), NS and sh3 lentivirus-transduced cells. Cell lysates were immunoprecipitated with anti-acetyllysine (AcK) antibody and immunoblotted with Ku70 antibody. Graph (lower panel) represents the densitometry quantification of mean Ku70 acetylation±S.D. of three independent experiments using QuantityOne software (VersaDoc imaging system, BioRad). (b) Sequential IP assay to check the in vivo association of SMAR1, Ku70 and HDAC6 in one complex. HCT116 cell lysate (1 mg) was first immunoprecipitated with SMAR1 antibody and eluate was subsequently probed with indicated antibodies, followed by second IP with HDAC6 antibody and immunoblotting with mentioned antibodies. (c) IP assay in NS and sh3 lentivirus-transduced HCT116 cells to investigate the association between Ku70 and HDAC6. Whole-cell lysates were immunoprecipitated with either Ku70 or HDAC6, and then immunoblotted with indicated antibodies. (d) In silico analysis of HDAC6–SMAR1–Ku70 complex. C-terminal of SMAR1 (green spheres) is sandwiched between Ku70 (magenta) and HDAC6 (cyan) (upper panel). Anterior and posterior view of the triple complex (lower panel). (e) Protein lysates extracted from control and tubacin-treated (10 μM, 2 h) HCT116 cells, which were previously transduced with either Ad-SM or sh3 lentivirus were analyzed for Ku70 acetylation by IP with anti-AcK antibody and immunoblotting for Ku70. (f) Western blot analysis to check IR-induced recruitment of SMAR1 and Ku70 to the chromatin in HCT116 cells that were previously transduced with either NS or sh3 lentivirus