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. 2017 Jul 22;7(12):3138–3154. doi: 10.7150/thno.19506

Figure 6.

Figure 6

RBFOX3 interacted with AP-2β and regulated the hTERT expression. (A) The extracted proteins from Hep3B and HepG2 cells with stable overexpression of flag-RBFOX3 were immunoprecipitated with antibody against KLF4 or RFPL3 or CPSF4 or AP-2β. The precipitates were analyzed by immunoblot using anti-flag antibody (left panel). The whole cell lysate (WCL) was analyzed by immunoblot (right panel). (B) The extracted proteins from Hep3B and HepG2 cells with stable overexpression of flag-RBFOX3 were immunoprecipitated with antibody against Flag. The precipitates were analyzed by immunoblot using anti-AP-2β antibody (left panel). The whole cell lysate (WCL) was analyzed by immunoblot (right panel). (C) Hep3B and HepG2 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of RBFOX3 with AP-2β were examined by confocal microscopy analysis. (D) Chromatin immunoprecipitation assays (ChIP) were done using antibody against AP-2β. The PCR products of hTERT promoter (-378 to -157) were separated on 2% agarose gels. (E) ChIP assays were carried out using the hTERT promoter primers and RBFOX3 antibody in Hep3B cells transfected with RBFOX3, RBFOX3 and siAP-2β, AP-2β, AP-2β and shRBFOX3, respectively. (F) Relative hTERT promoter activity in Hep3B cells transfected with RBFOX3, RBFOX3 and siAP-2β, AP-2β, AP-2β and shRBFOX3, respectively. (G) The expression of hTERT in Hep3B cells transfected with RBFOX3, RBFOX3 and siAP-2β, AP-2β, AP-2β and shRBFOX3, respectively. (H) MTS was performed in Hep3B cells transfected with RBFOX3, RBFOX3 and siAP-2β, AP-2β, AP-2β and shRBFOX3, respectively.