Figure 2
From: OTUD4 enhances TGFβ signalling through regulation of the TGFβ receptor complex
OTUD4 regulates TGFβ receptor activity. (A) HEK293T cells transfected with OTUD4 shRNA hairpins B and C. Cells were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (B) Quantification of pSMAD2 levels represented in (A). Data are shown as the mean ± SD of 3 independent experiments. *P < 0.05 as determined by Student’s T-Test. (C) HEK293T stimulated with TGFβ (100 pM) for 1 or 3 h. 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. (D) HEK293T cells transfected with FLAG-OTUD4, or FLAG OTUD4 DD (C45S-H148A). Cells were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (E) HEK293T cells transfected with FLAG-OTUD4 were stimulated where indicated with TGFβ (100 pM) for 1 h prior to SB431542 (1 µM). Cells were collected as indicated and whole cell extracts were probed with indicated antibodies. β-Tubulin is used as the loading control. (F) Spearman’s analysis between OTUD4 mRNA expression and TGFβ enrichment score (Hallmark geneset) score across a TCGA pan-cancer dataset (n = 12,290). Full-length blots for (A,D,E) are shown in Supplementary Information.
