Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: CtBP2 formed transcriptional complexes with p300 and c-Jun or c-FOS. (A) In vivo pull-down of the Flag-c-Jun- and c-FOS-associated complexes. RPTEC/TERT1 OAT3 cells were transfected with pCDNA3-2×Flag, pCDNA3-2×Flag-c-FOS or pCDNA3-2×Flag-c-Jun. After incubation for another 48 h, cells were subjected to immunoprecipitation analysis. The purified protein complexes were loaded onto SDS-PAGE gels, and protein bands were visualized using sliver staining. The IgG, c-Jun, c-FOS and CtBP2 bands are indicated. (B) Verification of the association of CtBP2, p300 and c-Jun or c-FOS in vivo . Protein samples used for mass spectrometry were applied to western blotting analyses to verify the association of CtBP2, p300 and c-Jun or c-FOS. (C) CtBP2 could not interact directly with c-Jun or c-FOS. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag-c-Jun; pCDNA3-6×Myc+pCDNA3-2×Flag-c-FOS; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-Jun; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-FOS. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures. (D) CtBP2 directly interacts with p300. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag; pCDNA3-6×Myc+pCDNA3-2×Flag-p300; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-p300. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures. (E) p300 directly interacted with c-Jun or c-FOS. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag-c-Jun; pCDNA3-6×Myc+pCDNA3-2×Flag-c-FOS; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-Jun; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-FOS. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: In Vivo, Transfection, Incubation, Immunoprecipitation, Purification, SDS Page, Staining, Mass Spectrometry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: Knockdown of CtBP2 caused the repression of TGFB1 . (A) The CtBP2 mRNA level. The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by RNA isolation and qRT-PCR analysis to measure CtBP2 mRNA levels. *** P < 0.001. (B) The TGFB1 mRNA level. RNA samples used in (A) were applied to qRT-PCR analysis to measure TGFB1 mRNA levels. ** P < 0.01 and *** P < 0.001. (C) Knockdown of CtBP2 inhibited TGF-β signaling. Cells used in (A) were subjected to protein isolation and western blotting to examine the protein levels of CtBP2, TGF-β, pSmad2, Smad2 and GAPDH. (D) Treatments with CtBP inhibitors could not change the mRNA level of CtBP2 . The RPTEC/TERT1 OAT3 cells were treated with 5 μM MTOB or NSC95397 for 4 h. Cells were then further treated with or without 50 ng/mL IL-1β for 2 h. The resulting cells were subjected to RNA isolation and qRT-PCR analysis to measure CtBP2 mRNA levels. (E) Treatments with CtBP inhibitors significantly repressed TGFB1 mRNA levels. RNA samples used in (D) were applied to qRT-PCR analysis to measure TGFB1 mRNA levels. *** P < 0.001. (F) Treatments with CtBP inhibitors inhibited TGF-β signaling. Cells used in (D) were subjected to protein isolation and western blotting to examine the protein levels of CtBP2, TGF-β, pSmad2, Smad2 and GAPDH.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: Knockdown, Transfection, Incubation, Isolation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: Knockdown or blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . (A) Knockdown of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P < 0.01 and *** P < 0.001. (B) Blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The human RPTEC/TERT1 OAT3 cells were treated with 5 μM MTOB or NSC95397 for 4 h. Cells were then further treated with or without 50 ng/mL IL-1β for 2 h. The resulting cells were subjected to ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P < 0.01 and *** P < 0.001.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: Knockdown, Transfection, Incubation