Expression of Concern: Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner (original) (raw)
Fig 1
Delineation of physical interaction between SATB1 and β-catenin.
(A) SATB1 and β-catenin colocalize in the thymocyte nuclei. Indirect immunofluorescence staining of thymocytes using antibodies to SATB1 (red) and β-catenin (green) was performed as described in Materials and Methods. DNA counterstaining was performed using DAPI (blue). The cut view panel depicts two perpendicular transverse sections of a triple-stained thymocyte as indicated by white lines, intersecting at the point of the brightest fluorescence signal. (B) Direct interaction between SATB1 and β-catenin was monitored by in vitro pulldown assays performed as described in Materials and Methods. 35S-labeled SATB1 was specifically pulled down after incubation with immobilized GST-β-catenin (lane 3) and not with control immobilized GST (lane 2). (C) In vivo interaction of SATB1 and bcatenin was assessed by performing coimmunoprecipitation analysis as described in Materials and Methods. Nuclear extracts derived from BIO treated (+) and control (-) human thymocytes were immunoprecipitated using anti-β-catenin followed by WB with anti-SATB1. (D) The interacting regions of SATB1 and β-catenin were mapped by in vitro pulldown assay. GST pulldowns of SATB1 and β-catenin were performed by passing Jurkat nuclear extract on immobilized domains of GST-β-catenin (lanes 1–6) and SW480 nuclear extract on immobilized domains of SATB1 (lanes 7–11) including both full-length proteins followed by WB with anti-SATB1 and anti- β-catenin. SATB1 and β-catenin truncations used are depicted schematically on the left. Solid black bars depict the respective interacting regions. (E) Coimmunoprecipitation analysis of extracts derived from HEK 293T cells overexpressing 3XFlag-SATB1 and its functional domains using anti-Flag antibody followed by WB with anti-β-catenin (lanes 1–3). Expression levels of the 3XFlag-fused domains of SATB1 were monitored by WB using anti-Flag (lanes 4–6). (F) Mammalian two hybrid assay was performed to score for protein-protein interactions in HEK 293T cells essentially as described [23]. The C-terminus of β-catenin and the PDZ-like domain of SATB1 were expressed as fusions with VP-16 and GAL4-DBD using the pACT and pBIND vectors of the CheckMate mammalian two-hybrid system (Promega). pBIND and pACT fusion constructs were transfected along with a reporter vector pG5-Luc containing 4xGAL4 responsive element and luciferase activity was compared with the control. Error bars represent standard deviation calculated from triplicates. (G) The C-terminus and not the N-terminus of β-catenin is involved in its interaction with SATB1. VP-16 fused C-terminal (aa 666–780, lane 1) and N-terminal (aa 1–137, lane 2) regions of β-catenin were overexpressed in HEK 293T cells. Co-immunoprecipitation was performed as described in Materials and Methods using anti-SATB1 followed by WB using anti-β-catenin.