Fig. 3From: Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinomaMethylation inhibits CLDN11 promoter activity by impairing the binding of the transcriptional activator GATA1. a Different promoter activities of a series of CLDN11 promoter deletion luciferase reporters were assayed in TW02 and HK1 cells. Intergroup comparison was conducted relative to the empty vector (pGL3). b The minimal CLDN11 promoter reporter (− 213 to + 197) and the FLAG-tagged GATA1 and GATA2 expression clones were cotransfected into TW02 and HK1 cells. Three putative GATA binding sites: GATA1 (− 92), GATA1/2 (− 62), and GATA2 (+ 184), are indicated. GATA1: GATA1 overexpression, GATA2: GATA2 overexpression, GATA1 + 2: GATA1 and GATA2 co-overexpression. Intergroup comparison was conducted relative to the vector control. The expression levels of ectopic GATA-1, GATA-2, and actin (internal control) were examined through Western blotting. c Minimal CLDN11 promoter reporters with wild-type (WT) and three mutated GATA binding sites (MU, S1–S3) were transfected into TW02 and HK1 cells, respectively. Intergroup comparison was conducted relative to the empty vector (pGL3). a~c All the luciferase reporter activities were normalized with respect to renilla activity. d DNA pull-down assay was used to analyze the binding affinity of exogenous FLAG-tagged GATAs (GATA1–3F and GATA2-3F) to WT, methylated (ME), and mutated (MT) biotinylated probes containing GATA1/2 site; the sequences of these probes are shown in the upper panel. Methylated cytosine is indicated by using “m” above C, and mutated sequences are underlined. Anti-GATA1 and anti-FLAG antibodies were used for examining the amount of bound GATA1–3F and GATA2-3F in the immunoprecipitates and 5% input. e EMSA was performed to compare the binding affinity of purified recombinant GATA1 for WT, ME, and MT biotinylated probes of the GATA1/2 site. The arrow indicates DNA–protein complexes. Antibodies against GATA1 were used for supershift assaysBack to article page