FIGURE 7.
HSP70 regulates NOD2 Crohn mutant activity. A, co-immunoprecipitation was performed in HEK293T cells transfected with 1 μg of pBKCMV, pBKCMV-NOD2/702Myc, pBKCMV-NOD2/908Myc, and pBKCMV-NOD2/1007insCMyc vectors, using 1 μg of mouse anti-Myc antibody per 150 μl of cell lysate. Eluates were probed for HSP70 using 1:1000 of rat anti-HSP70 and rabbit anti-Myc antibody. B, cell lysates from HEK293T cells transfected with 750 ng of pBKCMV/NOD2Myc, pBKCMV-NOD2/702Myc, pBKCMV-NOD2/908Myc, or pBKCMV-NOD2/1007insCMyc vectors along with 1 μg of HSP70 or control vector were probed using 1:1000 rabbit anti-Myc antibody and 1:1000 rabbit anti-HSP70 antibody. β-Actin was used as a loading control. Western blots were performed on separate cell lysates at least three times. Using the replicates, the bands were quantified using ImageLabTM. C, dual-luciferase assays were performed on HEK293T cells transfected with 50 ng of pBKCMV/NOD2Myc, pBKCMV-NOD2/702Myc, pBKCMV-NOD2/908Myc, or pBKCMV-NOD2/1007insCMyc vectors along with 50 ng of HSP70 or control vector/NOD2Myc and HCT 116 transfected with HSP70 or control vector in the presence or absence of 2 μm MDP. 10 ng of pGL432 and 1 ng of pRL vector were used per 24-well dish for transfection. Relative luciferase activity of firefly to Renilla is plotted. Results shown are the means ± S.D. of experiments performed in triplicates. *, p < 0.05 was considered as significant.