Fig. 1.

EGF induced the interaction between MIIP and RelA. a HCT116 cells were treated with or without EGF. Cellular nucleus-extracts subjected to immunoprecipitation with an anti-MIIP antibody. b HCT116 cells transfected with or without plasmid for expressing the indicated MIIP shRNA were treated with or without EGF for indicated periods of time. c, d HCT116 cells transfected with or without plasmid for expressing MIIP shRNA (c), and HCT116 cells expressed with wild type (WT) RelA and RelA K310R (d) were treated with or without EGF (100 ng/ml). Cell invasion assays were performed. e HCT116 cells expressed with WT RelA and RelA K310R were treated with or without EGF (100 ng/ml) for 10 h. Relative mRNA levels were analyzed by q-PCR. f HCT116 cells transfected with or without plasmid for expressing MIIP shRNA were treated with or without EGF (100 ng/ml) for 10 h. ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. g HCT116 cells were pretreated with Bis-I (2 μM), U0126 (20 μM) for 1 h, prior to EGF treatment (100 ng/ml) for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag antibody. h Purified GST-MIIP protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min. GST pull down analyses were performed (left panel). Purified GST–RelA protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min (right panel). In a, b, g, h, immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In c–f, the values are presented as mean ± s.e.m. (n = 3 independent experiments), * represents P < 0.05 and ** represents P < 0.01 (Student’s t-test) between the indicated groups