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. 2008 Mar 28;99(4):755–761. doi: 10.1111/j.1349-7006.2008.00746.x

Figure 3.

Figure 3

Domains relevant to the interaction between the Cnot1 scaffold protein and Tob. (a) Schematic diagram of Cnot1 and Tob expression constructs used in co‐immunoprecipitation assays. Cnot1(N), the amino terminal 1700 amino‐acid sequence of Cnot1; Cnot1(C), the carboxyl terminal 700 amino‐acid sequence of Cnot1; TobN113, amino terminal 113 amino‐acid sequence of Tob; TobC117, the carboxyl terminal 233 amino‐acid sequence of Tob. (b) The Flag‐tagged, full‐length Cnot1 (FL), Cnot1(N), or Cnot1(C) were exogenously expressed in COS7 cells with or without Myc‐tagged Tob. Proteins in the cell lysates were immunoprecipitated with anti‐Flag antibody, and the immunoprecipitates were separated by 7.5% (SDS‐PAGE) and subjected to Western blotting with the anti‐Myc (top panel) or anti‐Flag (middle panel) antibodies. Proteins in cell lysates were also separated by 7.5% SDS‐PAGE and subjected to Western blotting with the anti‐Myc antibody (bottom panel). (c) The Flag‐tagged, full‐length Tob, TobN113, or TobC114 were exogenously expressed in COS7 cells. Proteins in cell lysates were immunoprecipitated with anti‐Flag antibody. Western blot analyses of the cell lysates and the immunoprecipitates were carried out as in (b). (d) The Flag‐tagged, full‐length Cnot3, or Cnot6L in the pcDNA vector were exogenously expressed in COS7 cells with or without Myc‐tagged Tob. Proteins in the cell lysates were immunoprecipitated with anti‐Flag antibody. Western blot analyses of the cell lysates and the immunoprecipitates were carried out as in (b). (e) Schematic illustration of the interaction between Tob/BTG family proteins and the CCR4‐NOT complex. Subunits of the complex other than Cnot1 and Cnot7 are shown as white circles. Btg represents the family members except Tob. Tob is shown to be unique among the family proteins in that it likely interacts with the CCR4‐NOT complex through Cnot1 as well as through Cnot7.