Mobility shift of exogenous Borealin. Mobility shift and protein levels of exogenous Borealin in (A), (B), (C) and (D) were determined by Western blotting using an antibody to the Flag-tag. Transfers were stripped and reprobed for β-actin as a loading control. UT: untransfected. The multiple blocks in (A) are bands from the same gel. (A) Two electrophoretic forms of Borealin. Asynchronously growing Hela cells were transiently transfected with either wild type or S165A phosphomutant of Borealin. Transfected cells were either left untreated or treated with nocodazole for 14 hours to block cells in mitosis. Cell lysates were separated using 15% SDS-polyacrylamide gel. (B) Mobility shift of exogenous Borealin. Mitotic WT-8 cells were collected either by mitotic shake-off or by using nocodazole. Cell lysates were separated on a 12.6% SDS-polyacrylamide gel and analyzed by Western blotting. Lysates were less concentrated than those used in "A" and in asynchronous cells (No NOC) only the more abundant faster migrating form is visible. (C) Expression level of ectopic Borealin in different clones. Clones of Hela cells stably expressing different levels of wild-type Flag-Borealin were analyzed by Western blotting using 12.5% SDS-polyacrylamide gel. (D) Mobility shift of Borealin in WT-1. WT-1 cells stably expressing a lower level of wild-type Flag-Borealin than WT-8 were either left to grow asynchronously (No NOC) or blocked in mitosis using nocodazole (NOC). Cell lysates were separated on a 12.6% SDS-polyacrylamide gel. (E) Expression of GFP in Hela cells. Hela cells were transiently transfected with CMV-gfp and left to grow either asynchronously or treated with nocodazole. Cell lysates were separated by 12.5% SDS-polyacrylamide gel and subjected to Western blotting with anti-GFP antibody.
Kaur et al. BMC Cell Biology 2007 8:5 doi:10.1186/1471-2121-8-5