Figure 4

Effects of Tip110 immunodepletion and add-back on pre-mRNA splicing. Hela nuclear extract was pre-incubated with Tip110 mAb-conjugated protein-A beads to deplete endogenous Tip110 protein, as determined by Western blot analysis (a), and then used for pre-mRNA splicing reaction (b). An isotype-matched immunoglobulin control (IgG) was included as an immunodepletion control. Tip110-depleted nuclear extract was supplemented with recombinant Tip110 (100 ng) for the splicing reaction (b). Splicing was quantitated by densitometric scanning of the intronless spliced RNA product on the autoradiography, the spliced RNA product in the reaction containing the control IgG-immunodepleted Hela nuclear extract was set as the reference to calculate the relative level of pre-mRNA splicing (c).