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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Mol Cancer Res. 2018 Aug 2;16(12):1991–2002. doi: 10.1158/1541-7786.MCR-18-0366

Figure 6.

Figure 6.

Role of kinase pathways and proteasome complex proteins on SMAD7 ubiquitination and cell migration. A549 cells were transfected with FLAG-SMAD7 and treated with TGFβ (A), transfected with MKK4(WT) (B), transfected with MKK7(CA) or MKK7(DN) (± TGFβ) (C), and treated with various agents. Whole cell lysates were immunoprecipitated with FLAG antibodies and analyzed for ubiquitinated FLAG-SMAD7 by ubiquitin antibodies. A549 cells were transfected with FLAG-SMAD7, treated with TGFβ alone or in combination with oligonucleotides that knockdown Axin2, arkadia and RNF12 (D), treated with TGFβ alone or in combination with 14–22 Amide or siPKA-Cα (transfected) (E) or MKK4(WT)/MKK7(CA) (transfected) alone or in combination with 14–22 Amide or siPKA-Cα (transfected) (F), and whole cell lysates were immunoprecipitated FLAG antibodies and analyzed for ubiquitinated FLAG-SMAD7 using ubiquitin antibodies. (G) A549 cells were transfected with FLAG-SMAD7 and treated with TGFβ alone or in combination with 14–22 Amide or siPKA-Cα (transfected), immunoprecipitated with FLAG antibodies, and the immunoprecipitate was analyzed by a western blot. (H) A549 cells were treated with TGFβ and transfected with siAxin2, siArkadia and siRNF12 oligonucleotides and effects on cell migration were determined in a Boyden chamber assay. (I) The efficiency of siAxin2, siArkadia and siRNF12 on protein knockdown was determined by western blot analysis of whole cell lysates.