Skip to main content
. 2012 May 30;40(16):7676–7689. doi: 10.1093/nar/gks509

Figure 5.

Figure 5.

IKKα binds directly to RNAPII CTD S2p. (A) eCoIP performed with whole cell extract from RAW264.7 cells treated with LPS for 1 h. Antibodies used for immunoprecipitation are named at the top of the figure and antibodies used for western blot named on the left. Input was 5% of material used for CoIP. (B) In vitro pulldown followed by dot blot assay. GST-IKKα (2 pmol) was incubated in the absence of peptide (lane 2) or in the presence of 600 pmol CTD-S2p (lane 3) or CTD-S5p (lane 4) peptides or an equimolar mix of the two (lane 5). A GST-control peptide, not interacting with RNAPII, was incubated with the same equimolar mix of the two peptides (lane 1). Pulldown was performed with anti-GST beads. Unbound and bound factions were transferred onto the membrane. Antibodies used for dot blot are named on the left. (C) The competition between peptides for binding to IKKα was quantified using densitometry. Data are expressed as a ratio (bound/unbound)S2p + S5p/(bound/unbound)S2p or S5p. Error bars represent standard deviation (SD) of two independent experiments. (D) In vitro pull-down experiment performed with a biotinylated peptide corresponding to the CTD S2p incubated with recombinant HP1α, HP1γ and IKKα proteins in the presence or absence of ATP.