Proteomic analysis of Tip110-binding cellular proteins including YB-1. A. 293T cells were transfected with pTip110-HA. pcDNA3 was used in the mock transfection. Seventy-two hours post transfection, the cells were harvested for cell lysates. The lysates were applied to an anti-HA affinity matrix column, Tip110-binding proteins were then eluted from the column and analyzed on 10% SDS-PAGE followed by Coomassie blue staining. B. 293T cells were transfected with pTip110-HA, pYB-1-Myc or both and harvested 72 hr post transfection for cell lysates. pcDNA3 was added to equalize the total amount of transfected DNA. Cell lysates were directly used for Western blotting using anti-HA or anti-Myc antibody (top panels), or immunoprecipitated using anti-HA, followed by Western blotting using anti-HA or anti-Myc antibody (bottom panels). C. 293T cells were transfected with Tip110-HA and immunoprecipitation was performed using an anti-HA antibody or isotype-matched IgG, followed by Western blotting using anti-YB-1 or anti-HA antibody.
Timani et al. BMC Molecular Biology 2013 14:14 doi:10.1186/1471-2199-14-14