Interaction of +TIP protein EB1 with BubR1 for proper metaphase chromosome alignment
In mitosis accurate segregation of chromosome requires proper attachment of microtubule to kinetochore. Although many proteins are found to be involved in this process, the actual mechanism by which microtubule attach to kinetochore and the stabilization of this interaction is not fully understood. EB1 is a microtubule + end binding protein, which is involved in kinetochore microtubule association and stabilization of this interaction. It is found to be present at the microtubule, and in the surrounding of outer kinetochore, during metaphase. It’s known, EB1 depletion can affect the chromosome congression without the initiation of spindle assembly checkpoint …show more content…
In EB1 immunoprecipitate immunoblotting analysis identified BubR1.To verify the interaction between EB1 and BubR1 further, I performed a reverse coimmunoprecipitation using BubR1 antibody from HeLa cell lysate. Immunoblotting analysis showed EB1 in BubR1 immunoprecipitate. To conform the results, I did a GST Pull down using purified EB1-GST, EB1C-GST, GST proteins from HeLa cell lysate. For this, first I incubated purified EB1-GST, EB1C-GST and GST proteins with HeLa cell lysate and pulled down the GST proteins with glutathione beads. Immunoblotting analyses of precipitate showed, BubR1 was pulled down by EB1-GST and EB1C-GST proteins and not by GST protein. This results not only shows EB1 is interacting with BubR1, but also C terminal of EB1 is interacting with BubR1. Further analysis using immunoblotting showed Mad2 is also present in the immunoprecipitate. To find whether EB1 works together with BubR1 to regulate spindle-kinetochore attachment, I examined the localization of BubR1 and EB1 in metaphase of HeLa cells using confocal microscope, and I found during metaphase EB1 and BubR1 colocalize at the outer kinetochore