Fig. 3

Co-immunoprecipitation of Myc-SCAF4-CID with recombinant CTD. a A diagram of mammalian expression constructs expressing GST-CTD and Myc-fusion proteins. b Total lysates from HEK293T cells transfected with GST (lane 1), GST-CTD (lane 2), Myc-SCAF4-CID (lane 3), Myc-p72b (lane 4) or the indicated combinations (lanes 5–8) were immunoblotted with anti-GST or anti-Myc to detect the expression of GST-CTD or the Myc-fusions. c Cell lysates as in (a) were immunoprecipitated with anti-Myc (9E10) and probed with anti-GST. Total lysates from co-transfected samples (lanes 5–8) were also immunoprecipitated with mouse IgG (lanes 9–12) and probed with anti-GST. d Binding of Myc-SCAF4-CID to biotin-pS₂-CTD but not biotin-pY₁pS₂-CTD peptides. The indicated amount of each biotinylated CTD-peptide was reacted with 50 µg of total lysates from HEK293T cells transfected with the Myc-SCAF4-CID expression plasmid and then pulled down by streptavidin beads. Values shown in the upper panel are mean and standard deviation of densitometry quantification of anti-Myc immunoblotting for the Myc-SCAF4-CID in the pull-down fraction from three independent experiments. Representative blots of total lysate (input) and the pull-down fractions are shown in the lower panel