Repression domain 3 of PUM1&2 binds directly to the CNOT complex. (A) Coimmunoprecipitation of RD3 domains of PUM1&2 proteins from HCT116 cells. V5-CNOT8 served as a positive control and empty vector served as a negative control. Western blot of each V5-tagged bait protein in cell extracts (Input) and anti-V5 immunoprecipitates (IP). Endogenous CNOT subunits were detected by western blot. Cell extracts were treated with RNases A and 1, and RNA digestion was confirmed in Supplemental Figure S4. (B) Coomassie stained SDS-PAGE of in vitro pulldown assays with recombinant purified RD1, RD2, RD3, and RBD domains of PUM1 and PUM2, produced as fusions with maltose binding protein (MBP) and StrepII (strep) affinity tags with the intact purified CNOT complex (Input). MBP-strep served as negative control. (C) Same as B, but with the four indicated CNOT modules (Input). Diagram of CNOT modules is shown in Figure 1B.
