Figure 1
From: OTUD4 enhances TGFβ signalling through regulation of the TGFβ receptor complex
OTUD4 activates the canonical TGFβ pathway. (A) TGFβ responsive luciferase (CAGA luciferase) of HEK293T cells transfected with four independent OTUD4 shRNA hairpins labelled A, B, C and D. Cells were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Error bars represent SD of triplicates. Experiment is a representative of 3 independent experiments. ***P ≤ 0.001 as determined by Student’s T-Test. (B) Western blot analysis of HEK23T cells transfected with FLAG-OTUD4 and OTUD4 knockdown shRNA hairpins A, B, C and D. β-Actin is used as the loading control. (C) Western blot analysis of HEK293T cells transfected with OTUD4 knockdown shRNA hairpins B and C. Immunoblotting for OTUD4 was performed. β-Actin is used as the loading control. (D) HEK293T cells were transfected with OTUD4 knockdown constructs B and C or relevant controls. OTUD4 mRNA levels relative to GAPDH are shown as evaluated by quantitative real-time PCR. Data are shown as the mean ± SD of triplicate samples from a representative experiment performed three times. (E). HEK293T OTUD4KD1 cells were stimulated with TGFβ (100 pM) for 3 h. SMAD7, CTGF, PAI1 and OTUD4 mRNA levels relative to GAPDH are shown as evaluated by quantitative real-time PCR. Data are shown as the mean ± SD of triplicate samples from a representative experiment performed three times. ***P ≤ 0.001 as determined by Student’s T-Test. (F) TGFβ responsive luciferase (CAGA luciferase) of HEK293T cells transfected with FLAG-OTUD4 WT, C45S, H148A, D42A, or C45S-H148A. Cells were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Data are shown as the mean ± SD of triplicate samples from a representative experiment performed three times. ***P ≤ 0.001 as determined by Student’s T-Test. Full-length blots for (B,C) are shown in Supplementary Information.
