Figure 3: TGF-β upregulates PHF14 expression through the TGF-β/phosphorylated smad2/3 pathway. | Scientific Reports

Figure 3: TGF-β upregulates PHF14 expression through the TGF-β/phosphorylated smad2/3 pathway.

From: PHF14: an innate inhibitor against the progression of renal fibrosis following folic acid-induced kidney injury

Figure 3

NRK-49F cells were pretreated with the TGF-β receptor 1 inhibitor SB431542 (0.6 μM) or serum-free medium for 30 min before TGF-β stimulation. (A) The schematic diagram of the PHF14 promoter region and the primer design. The region from -528 bp to -519 bp is the potential binding site of the smad3/4 complex predicted by virtual laboratory PROMO. (B) Quantitative PCR (Q-PCR) analysis showing that the otherwise elevated PHF14 mRNA level under TGF-β stimulation in the presence of SB431542 was suppressed. *P < 0.05, compared with the PHF14 mRNA level at baseline (0 h) (n = 3). (C) Western blot analysis demonstrating the PHF14 expression level under TGF-β stimulation in cells pretreated with SB431542, compared with NRK-49F cells pretreated with serum-free medium. Anti-GAPDH was used to verify equivalent loading. (D) Semiquantitative analysis of PHF14 protein abundance. *P < 0.05, compared with scrambled siRNA controls (n = 3). (E) Chromatin immunoprecipitation of the PHF14 promoter region with anti-p-smad3 antibody. P1–P4, primers 1 to 4; TSS, transcription start site; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF-β, transforming growth factor-β; NC, negative control; PC, positive control.

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