Fig. 4.

MIIP prevent HDAC6-mediated RelA deacetylation. a HCT116 cells were pretreated with or without 4SC-202 (0.6 μM), FK228(20 nM) and Nexturastat A (5 nM) for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Immunoblotting analyses were performed. b HCT116 cells were pretreated with or without Bis-l for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag, followed by Flag-beads washing and a second immunoprecipitation with an anti-MIIP (lanes 1–4 from left). Cellular extracts subjected to immunoprecipitation with an anti-Flag (lanes 5–8 from left). c–e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6 shRNA; cells were treated with or without EGF (100 ng/ml) for 10 h. Immunoblotting analyses were performed (c). ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of Twist or MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. d Relative mRNA levels were analyzed by q-PCR (e). In a–c, immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In d, e, the values are presented as mean ± s.e.m. (n = 3 independent experiments), ** represents P < 0.01 (Student’s t-test) between the indicated groups