Pcdna3.0 vector

For the study of the subcellular localization and coimmunoprecipitation (Co-IP) test between bfACP3 and bfTRAF6, full-length bfACP3 was inserted into the pcDNA3.0 vector (Clontech) with a C-terminal HA Tag (transformed by our laboratory, the HA coding sequence was inserted after the XbaI restriction site) and bfTRAF6 was fused with Flag tag and inserted into the pcDNA 3.0 vector (Clontech, transformed by our laboratory, the Flag coding sequence was inserted in front of the Kpn I restriction site). For the expression of the truncated mutants of bfACP3, PCR fragments encoding amino acids 24-561, 24-204, 205-561, 205-358, 165-358, 124-358, 24-358 and 359-561 were fused with myc tag, and inserted into the expression plasmid pCMV-Myc vector (Clontech). For the reporter assays and ubiquitination experiment, full-length bfACP3 was cloned into the pCMV-Myc vector (Clontech) and bfMyD88 was constructed in the same way as bfTRAF6. The full-length sequences of bfMyD88 and bfTRAF6 are shown in the Supplementary Figures 6 and 7, respectively. The ClonExpress^® II Kit (Vazyme) was used for the construction of recombinant expression vectors. The vectors were verified by sequencing and the expression of proteins were confirmed by western blot. Primers were described in Table 1.

Human HL7702 normal liver cells were maintained in RPMI‐1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin‐streptomycin (Gibco) at 37°C in 5% CO₂. For overexpression of miR‐194, a miR‐194 mimic (Ambion, Austin, USA) was added to complexes at a final concentration of 60 nmol/L. For miR‐194 knockdown, antisense oligonucleotides (Ambion) were added to complexes at a final concentration of 100 nmol/L. The mirVana™ miRNA mimic negative control (Ambion) was applied as a control. For ACVR2B overexpression, ACVR2B cDNA was amplified and cloned into the pcDNA3.0 vector (Clontech, Palo Alto, USA). Transfections were performed using a Lipofectamine 2000 kit (Life Technologies, Carlsbad, USA) according to the manufacturer's instructions. Transfected cells were harvested at 24 or 48 hours.