Figure 4. 14-3-3ζ overexpression led to TGFβ/Smads pathway activation in MCF10A cells via increasing TβRI expression.

(A) Immunoblot and RT-PCR analysis of TβRI and TβRII expression in MCF10A sublines.

(B) Immunoblot analysis of nuclear p-smads (p-smad2/p-smad3), T-smad2/3, with lamin C as nuclear fraction loading control.

(C) Chromatin immunoprecipitation assay with the indicated antibodies showed smad3 binding with ZFHX1B promoter in 10A.ErbB2.ζ and 10A.14-3-3ζ cells (arrows), not in 10A.Vec and 10A.ErbB2 cells (empty arrows).

(D) Knockdown of 14-3-3ζ by siRNA reduced TβRI and ZFHX1B expression in 10A.ErbB2.ζ and 10A.14-3-3 ζ cells. TβRI level and ZFHX1B level were analyzed by immunoblot and RT-PCR, respectively, 48 hours after siRNA transfection.

(E) 14-3-3ζ inhibited TβRI ubiquitination. 10A.ErbB2.ζ and 10A.ErbB2 cells were co-transfected with vectors expressing myc-TβRI and HA-ubiquitin (left), 10A.ErbB2.ζ and Hela cells were co-transfected with myc-TβRI, HA-ubiquitin, and control/14-3-3ζ siRNA (middle), Hela cells were co-transfected with myc-TβRI, HA-ubiquitin, and pcDNA3.Vec/pcDNA3.14-3-3ζ (right).After 48 hours, cell lysates were collected and subjected to immunoprecipitation and immunoblot with myc and HA antibodies.

(F) 10A.ErbB2 and 10A.ErbB2.ζ cells were treated with DMSO or 20 μg/ml MG132 for 6 hours; TβRI levels were analyzed by immunoblot.

(G) 14-3-3ζ associated with TβRI. 10A.ErbB2.ζ and 10A.14-3-3ζ cell lysates were immunoprecipitated by anti-HA antibody, followed by immunoblot analysis of TβRI.

(H) Schematic representation of different TβRI mutants.

(I) 14-3-3ζ binds to TβRI at its kinase domain between amino acid 210 and 370. 10A.ErbB2.ζ cells were infected with lentivirus expressing different TβRI mutants as in (H). Then the cell lysates were subjected to pull-down assay with GST or GST-14-3-3ζ, followed by immunoblot with TβRI antibody.