Research Paper Volume 8, Issue 10 pp 2308—2323

Figure 7. SIRT6 interacts with p27 in vivo and in vitro. (A) Co-IP of endogenous SIRT6 and p27 was performed in 293 cells. (B) Co-IP of exogenous SIRT6 (flag-SIRT6) and p27 (myc-p27) was performed using flag antibody in 293 cells. (C) 293 cells were transfected with flag-SIRT6 and p27, then immunofluorescence assay was carried out using flag and p27 antibodies to investigate the co-localization of these two proteins. (D) GST-pulldown assay using in vitro transcribed and translated SIRT6 or p27 and purified GST-p27 or GST-SIRT6 from E.coli BL21 cells. Blots were evaluated with SIRT6, p27 and GST antibodies. (E) Schematic representation of full-length p27 and its deletions. (F) 293 cells were transfected with flag-SIRT6 and flag-ΔSIRT6. Cell lysates were then used for co-IP with the flag antibody. Blots were evaluated with flag and p27 antibody. (G). 293 cells were transfected with flag-SIRT6 and myc-p27N or myc-p27C, co-IP was then performed using anti-myc antibody. Blots were evaluated with myc and SIRT6 antibodies.