Figure 3

Protein coimmunoprecipitation and competitive binding of Hinderin to SMC3. A) To monitor the Hinderin interaction, 293 cells were transfected with 1 μg/ml of Hinderin-V5 expression vector and incubated for 48 h. Mock transfected cells served as control. For SMC1 and SMC3 coimmunoprecipitation experiments, cell lysates (500 μl) were incubated with either 25 μg of anti-SMC1 or anti-SMC3 antibody for 2 h at RT. The immunocomplexes were then captured on agarose-protein G and analyzed by electrophoresis on 12% SDS-PAGE. After transblotting to a nitrocellulose membrane, Hinderin-V5 was identified using an anti-V5 monoclonal antibody and HRP-conjugated secondary antibody. The migration position of the Hinderin-V5 fusion protein present in the input cell lysate (200 μl) and in the immunoprecipitate is indicated by an arrow. Goat immunoglobulins are identified by an asterix. The presence of SMC1 and SMC3 in the Hinderin-V5 immunoprecipitate was assessed by incubation of cell lysates (500 μl) with 10 μg murine anti-V5 antibody. The immunocomplexes absorbed on agarose-protein G were analyzed on 8% SDS-PAGE and immunoblotted with either anti-SMC1 or anti-SMC3 antibody. SMC1 and SMC3 immunoblots of the input material (200 μl) are also illustrated. B) The effect of Hinderin on the interaction between the SMC3 and SMC1 hinge domains was investigated in 293 cells expressing different level of Hinderin by using a mammalian two-hybrid assay system. SMC3-474/702 and the SMC1-474/663 hinge domains in pBIND and pACT vectors respectively, were cotransfected in 293 cells together with Hinderin-V5 expression vector (0.3 or 1 μg/ml) or alternatively 1 μg/ml of the empty pcDNA3.1 vector (mock-transfected) and the reporter pG5/luc vector using Lipofectamine. Cells transfected with pBIND-Id and pACT-MyoD fusion protein expression vectors were used as control to assess the specificity of the Hinderin effect on protein-protein interaction. Luciferase activity was assayed after 48 h. The bars represent the mean ± SD of the values (n = 3). Semiquantitative RT-PCR was employed to assess the transcript level of GAL4:SMC3 and VP16:SMC1 fusion proteins and of G3PDH in cells ectopically expressing different levels of Hinderin.