Figure 4
Figure 4. The Trib2 COP1-binding site and COP1 are required for C/EBP-α degradation. (A) Interaction of Trib2 and COP1. 293T cells were transfected with COP1-HA (lane 1), myc-tagged Trib2-FL (lane 2), COP1-HA and myc-tagged Trib2-FL (lane 3), myc-tagged dCOP (lane 4), COP1-HA and myc-tagged dCOP (lane 5), myc-tagged VPM (lane 6), COP1-HA and myc-tagged VPM (lane 7), myc-tagged KDM (lane 8), COP1-HA and myc-tagged KDM (lane 9), myc-tagged K177R (lane 10), or COP1-HA and myc-tagged K177R (lane 11). Proteins were immunoprecipitated using the Myc 9E10 antibody, and Western blotting was performed with HA (top panels) and Myc (bottom panels) antibodies on immunoprecipitates (IP), and on whole cell lysates (WCLs). (B) COP1 knockdown decreases COP1 expression. Left panel, 293T cells were transfected with scrambled shRNA, or 1 of 2 different shCOP1 plasmids alone or together with COP1-HA. Western blotting was performed with HA antibody to detect COP1 expression. β-actin served as the loading control. Middle panel, 293T cells were cotransfected with COP1-HA and either the scrambled shRNA or each of the shCOP1 plasmids and analyzed by real-time RT-PCR for COP1 expression. Error bars denote SEM of each sample measured in triplicate. Right panel, sorted GFP+ 32D cells transduced with either shCOP1 or scrambled shRNA-expressing retrovirus were analyzed for endogenous COP1 knockdown by real-time RT-PCR. Error bars denote SEM of each sample measured in triplicate. (C) COP1 knockdown impairs the effects of Trib2 on C/EBP-α expression. Sorted GFP+ tNGFR+ 32D cells transduced with the indicated retroviral constructs were assessed for C/EBP-α protein expression by Western blot. The shRNA constructs were expressed from the low-molecular-mass polypeptide retroviral vector, which expresses GFP as a surrogate marker, whereas Trib2 was expressed from a version of MigR1 that expresses truncated NGFR as a surrogate marker.37 The cells that were not transduced with FL-Trib2 were transduced with the empty NGFR vector (lanes 2, 4, and 6). β-Actin was the protein loading control.

The Trib2 COP1-binding site and COP1 are required for C/EBP-α degradation. (A) Interaction of Trib2 and COP1. 293T cells were transfected with COP1-HA (lane 1), myc-tagged Trib2-FL (lane 2), COP1-HA and myc-tagged Trib2-FL (lane 3), myc-tagged dCOP (lane 4), COP1-HA and myc-tagged dCOP (lane 5), myc-tagged VPM (lane 6), COP1-HA and myc-tagged VPM (lane 7), myc-tagged KDM (lane 8), COP1-HA and myc-tagged KDM (lane 9), myc-tagged K177R (lane 10), or COP1-HA and myc-tagged K177R (lane 11). Proteins were immunoprecipitated using the Myc 9E10 antibody, and Western blotting was performed with HA (top panels) and Myc (bottom panels) antibodies on immunoprecipitates (IP), and on whole cell lysates (WCLs). (B) COP1 knockdown decreases COP1 expression. Left panel, 293T cells were transfected with scrambled shRNA, or 1 of 2 different shCOP1 plasmids alone or together with COP1-HA. Western blotting was performed with HA antibody to detect COP1 expression. β-actin served as the loading control. Middle panel, 293T cells were cotransfected with COP1-HA and either the scrambled shRNA or each of the shCOP1 plasmids and analyzed by real-time RT-PCR for COP1 expression. Error bars denote SEM of each sample measured in triplicate. Right panel, sorted GFP+ 32D cells transduced with either shCOP1 or scrambled shRNA-expressing retrovirus were analyzed for endogenous COP1 knockdown by real-time RT-PCR. Error bars denote SEM of each sample measured in triplicate. (C) COP1 knockdown impairs the effects of Trib2 on C/EBP-α expression. Sorted GFP+ tNGFR+ 32D cells transduced with the indicated retroviral constructs were assessed for C/EBP-α protein expression by Western blot. The shRNA constructs were expressed from the low-molecular-mass polypeptide retroviral vector, which expresses GFP as a surrogate marker, whereas Trib2 was expressed from a version of MigR1 that expresses truncated NGFR as a surrogate marker.37  The cells that were not transduced with FL-Trib2 were transduced with the empty NGFR vector (lanes 2, 4, and 6). β-Actin was the protein loading control.

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