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. 2015 Feb 27;11(4):434–447. doi: 10.7150/ijbs.9311

Figure 5.

Figure 5

TTC9A down-regulates ERα transcriptional activity in breast cancer cells. (A, B) ERE-luciferase assay showing a significant decrease in ERα transcriptional activity by TTC9A (25ng) and up-regulation in ERα transcriptional activity with TTC9A siRNA knockdown in MCF-7. Western blot showing that endogenous ERα expression level was not affected by TTC9A over-expression, and TTC9A protein level was drastically reduced by knockdown. Result shown (mean±SEM (n = 3) is representative of two independent experiments. The p values were obtained by Student's t-test. **p<0.01. (C) ERE-luciferase assay showing the effects of FKBP38 and FKBP51 (50ng) on ERα's transcriptional activity in MCF-7 cells. Western blot showing that endogenous ERα expression level was not affected by FKBP over-expression. Luciferase reporter assay result (mean±SEM (n = 3) shown is representative of two independent experiments *p<0.05, **p<0.01. (D) FKBP38 and FKBP51 interact with ERα and Hsp90. COS7 cells were transfected with ERα alone or together with FLAG-FKBP38 or FLAG-FKBP51. Anti-FLAG antibody pulled down both FKBPs and co-immunoprecipitated both ERα and Hsp90. ERα and Hsp90 were detected using anti-ERα and anti-Hsp90 antibodies respectively. FKBP38 and 51 were detected by anti-FLAG antibody. (E) TTC9A does not interact with ERα and Hsp90. COS7 cells were transfected with pXJ-FLAG control vector with ERα or FLAG-TTC9 with ERα. Anti-FLAG antibody pulled down FLAG-TTC9, but no ERα or endogenous Hsp90 proteins were co-immunoprecipitated. (F) TTC9A interacts with FKBP38 and FKBP51. COS7 cells were transfected with GFP-TTC9A and FLAG-FKBP38 or FLAG-FKBP51. Anti-FLAG antibody pulled down both FKBPs and co-immunoprecipitated TTC9A and Hsp90 at the same time. (G) TTC9A interacts with endogenous FKBP51. HeLa cells were transfected with FLAG-TTC9A or control vector and treated with 10 nM dexamethasone to induce the endogenous FKBP51. Anti-FLAG antibody was used to pull-down FLAG-TTC9A in the whole cell lysates and the co-immunoprecipitated FKBP51 was detected by anti-FKBP51 antibody.