Fig 1: TRADD-dependent caspase-8 activation does not cleave RIP1/RIP3. (A) WT and RIP1 KO cells were treated with TNF and then were analyzed by Western blot. The 2 h treated cells were imaged by scanning electronic microscopy (SEM). C8 (p43): p43 fragment of caspase-8. (B) Schematic overview of Mechanism 4 in Model 4. (C) Deviation (R2) between simulation results of Model 4 and SWATH-MS data. (D) Comparison between Model 4 predictions and experimental data of p-RIP3 regulated by RIP1 level (left panel), C8 activation in RIP1 KO and RIP3 KO cells (right panel). Western blot data can be found in Fig. S5A. (E) Simulation of RIP1 decrease induced increase of C8 upon TNF treatment. (F) RIP1 KD cells were treated with TNF and then were analyzed by Western blot. Pro-C3: pro-caspase-3; C3 (p17): p17 fragment of caspase-3. (G) Simulated effects of TRADD reduction on RIP1 decrease induced increase of C8. (H) RIP1 was KD in WT and TRADD KO cells. The cells were then treated with TNF and were analyzed by Western blot. (I) Comparison between Model 4 predictions and experimental data of RIP1 level mediated cell death rate at 4 h and 6 h TNF treatment. Expression levels of RIP1 KD cells were quantified from Fig. 3F
Fig 2: Effects of TRADD overexpression on the LPS-induced inflammatory responses and apoptosis of THP-1 cells. (A) Protein expression of TRADD was assessed after transfection with TRADD overexpression vector with or without miR-1184 mimic/miR-NC. ***P<0.001 vs. pcDNA-NC; ##P<0.01 vs. pcDNA-TRADD + miR-NC. (B) The expression levels of IL-6, TNF-a and IL-1ß in the miR-1184 mimic-transfected cells were significantly elevated after transfection with TRADD overexpression vector. (C) Flow cytometry was performed to estimate cell apoptosis in THP-1 cells transfected with miR-1184 mimic/miR-NC with or without TRADD overexpression vector. Data are expressed as mean ± SD (n=3). ###P<0.001 vs. LPS + miR-NC; ??P<0.01, ???P<0.001 vs. LPS + miR-1184 mimic+ pcDNA-NC. TRADD, TNF receptor type 1-associated DEATH domain protein; LPS, lipopolysaccharide; miR, microRNA; NC, negative control; IL, interleukin; TNF, tumor necrosis factor.
Fig 3: NF-?B signaling participates in the regulatory effect of miR-1184/TRADD in LPS-induced THP-1 cells. The protein levels of TRADD, p65 and p-p65 were detected by western blot analysis under LPS treatment after transfection with miR-1184 mimic/miR-NC with or without pcDNA-TRADD/pcDNA-NC. Data are expressed as mean ± SD (n=3). ***P<0.001 vs. control + miR-NC; ###P<0.001 vs. LPS + miR-NC; ?P<0.05, ???P<0.01 vs. LPS + miR-1184 mimic + pcDNA-NC. miR, microRNA; TRADD, TNF receptor type 1-associated DEATH domain protein; LPS, lipopolysaccharide; NC, negative control; p, phosphorylated.
Fig 4: Co-expression of A20/tumor necrosis factor receptor 1-associated death domain in the intestinal epithelium of mice across groups. A: Wild-type mice in normal control group; B: Wild-type mice in model control group; C: Wild-type mice in mesalazine group; D: Wild-type mice in herbs-partitioned moxibustion group; E: A20IEC-KO mice in normal control group; F: A20IEC-KO mice in model control group; G: A20IEC-KO mice in mesalazine group; H: A20IEC-KO mice in herbs-partitioned moxibustion group. Data are presented as the mean ± standard deviation (n = 10). Data were evaluated for statistical significance using one-way analysis of variance and are represented as follows: aP < 0.05, bP < 0.01 as compared to normal contro; cP < 0.05, dP < 0.01 as compared to model control; eP < 0.05, fP < 0.01 as compared to mesalazine; gP < 0.05, hP < 0.01 as compared to wild type. TRADD: Tumor necrosis factor receptor 1-associated death domain; WT: Wild type; NC: Normal control; MC: Model control; MESA: Mesalazine; HPM: Herbs-partitioned moxibustion.
Fig 5: Expression levels of A20, tumor necrosis factor alpha, tumor necrosis factor receptor 1, tumor necrosis factor receptor 1-associated death domain, receptor-interacting protein 1, and FAS-associated death domain protein across groups. Data are presented as the mean ± standard deviation (n = 10). Data were evaluated for statistical significance using one-way analysis of variance and are represented as follows: aP < 0.05, bP < 0.01 as compared to normal contro; cP < 0.05, dP < 0.01 as compared to model control; eP < 0.05, fP < 0.01 as compared to mesalazine; gP < 0.05, hP < 0.01 as compared to wild type. TNFR1: Tumor necrosis factor receptor 1; RIP1: Receptor-interacting protein 1; TNF-a: Tumor necrosis factor alpha; TRADD: Tumor necrosis factor receptor 1-associated death domain; FADD: FAS-associated death domain; WT: Wild type; NC: Normal control; MC: Model control; MESA: Mesalazine; HPM: Herbs-partitioned moxibustion.
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