Figure 4. Analysis of proteins co-precipitating with human CNOT7 after inactivation of individual subunits of the CCR4-NOT complex by siRNA treatment. HEK293 cells expressing stably CNOT7-TAP were transfected first with the indicated siRNAs and re-transfected 24 h later with the same siRNAs and a plasmid expressing GFP-CNOT6 (because no high quality commercial antibodies against CNOT6 or CNOT6L were available). Two days later, the cells were lysed and the CNOT7-TAP protein was precipitated with IgG sepharose beads. Co-precipitation of the CNOT proteins indicated on the right side of the figure was detected by western blotting (WB). A fraction corresponding to 10% of the input fraction was also analyzed by western blotting to monitor the level of individual subunit before precipitation. To assess reproducibility, each siRNA treatment was performed in duplicate. An anti-actin western blot was also performed to verify equal loading of the input samples.