Figure 4.

Sub1 associates with the Rpb4/7 heterodimer via direct interaction with Rpb7. (A) Pull down assay. 15% SDS-PAGE gel showing bands corresponding to recombinant proteins rSub1 and rRpb4/Rpb7-6His. (lanes 2 and 3, respectively) and pull down assays where rSub1 and a Co²⁺ resin have been incubated in the absence (lane 4) and in the presence of rRpb4/Rpb7-6His (lane 5). (B) Co-IP performed with IgG Sepharose using WCEs from wt strains expressing Sub1–TAP and Rpb7–HA/Rpb4–HA and from rpb4Δ cells expressing Sub1–TAP and Rpb7–HA. Input and IPs were analyzed by Western blotting with antibodies to the indicated proteins. (C) Co-IP performed on WCEs from Sub1–TAP cells (wt and rpb4Δ) using anti-Rpb3 antibody. As a control, the same amount of cell extracts was incubated only with Protein A sepharose. Inputs and IPs were analyzed by Western blotting with antibodies to the indicated proteins.