Fig. 9
From: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation
Difopein disrupts Eag1 interaction with Cul7. A GST pull-down assay of the interaction of Cul7 with Eag1 C-terminal region. Shown on the top is the structural topology for Eag1, as well as two GST-Eag1 C-terminal fusion proteins, GST-Eag1-C1-A and GST-Eag1-C1-B. (Left) Cell lysates prepared from HEK293T cells expressing Myc-Cul7 were used for pull-down assay with GST, GST-Eag1-C1-A, or GST-Eag1-C1-B, followed by immunoblotting with the anti-Cul7 or anti-GST antibodies. (Center) Myc-Cul7 was co-expressed with either YFP-R18 mutant or YFP-difopein in HEK293T cells, followed by pull-down assay with GST-Eag1-C1-B. (Right) Quantification of the relative pull-down efficiency. Protein densities were standardized as the ratio of Cul7 pull-down signals to the corresponding input signals, followed by normalization with respect to the YFP-R18 mutant co-expression control (*, p < 0.05; n = 3). B GST pull-down assay of the interaction of Cul7 with Eag1 N-terminal region. Shown on the top is the structural topology for the N-terminal fusion protein GST-Eag1-N. (Left) Cell lysates prepared from HEK293T cells expressing Myc-Cul7 were used for GST pull-down assay with GST or GST-Eag1-N. (Center) Myc-Cul7 was pulled down with GST-Eag1-N in the presence of either YFP-R18 mutant or YFP-difopein. (Rght) Quantification of the relative pull-down efficiency (*, p < 0.05; n = 3). C The effect of difopein on the co-immunoprecipitation efficiency of Cul7 and Eag1 in HEK293T cells. (Left) Representative immunoblots. Myc-Cul7, Eag1, and YFP-R18 mutant/YFP-difopein were co-expressed in HEK293T cells. 24 h after transfection, cells were treated with 10 μM MG132 for 12 h. Cell lysates were immunoprecipitated (IP) with the anti-Cul7 antibody, followed by immunoblotting analyses. (Right) Quantification of relative co-immunoprecipitation efficiency of Cul7 and Eag1. Protein densities were standardized as the ratio of Eag1 IP signals to the corresponding input signals, followed by normalization with respect to the YFP-R18 mutant co-expression control (*, p < 0.05; n = 3)