Pcdna3.1 v5 his

For forced expression experiments, cDNA encoding for ADAM32 was amplified by PCR using Fast Start Taq DNA Polymerase (Roche, Basel, Switzerland). The amplified fragment was cloned into pcDNA3.1/V5-His (Invitrogen) and p3xFLAG-CMV™-10 Expression Vectors (Sigma-Aldrich, St. Louis, MO, USA). For the Tet-inducible experiment, the region encoding 3xFLAG-ADAM32 (aa20 to aa787) was subcloned into pRetroX-Tight-Pur (Clontech, Mountain View, CA, USA).
For knockdown experiments, pSUPERIOR-puro (OligoEngine, Seattle, WA, USA) was used. Target sequences of shRNA were designed by using siDirect version 2 (http://sidirect2.rnai.jp, accessed on 27 July 2017) according to an established protocol [30 (link)]. The sense and antisense oligos for the target gene were annealed and subcloned into the pSUPERIOR-puro vector according to the manufacturer’s protocol. Table S2 shows the oligo sets and shRNA target sequences used. All constructs were confirmed by sequencing analysis. The shRNA vectors targeting the ADAM32 gene and LacZ are referred to as shADAM32 and shLacZ, respectively. The vector expressing mutant type TP53 (R248W) was constructed in a previous study [31 (link)] and is referred to as pCMX-p53-R248W.

Using the designed primers to remove translation termination codon (Table 1), the full-length FBXO7 cDNA fragments from an individual heterozygous for Cys52 were cloned into pGEM-T Easy vector (Promega) and sequenced. The 1.7 kb HindIII (added in the forward primer)–AgeI (added in the reverse primer) fragments were removed from pGEM-T Easy vector and ligated into the corresponding sites of pEGFP-N1 (Clontech) to generate Tyr52 and Cys52 FBXO7 cDNA in-frame fused to the EGFP gene. The resulting EGFP-tagged FBXO7 constructs were used in transient expression studies for confocal microscopy examination, FBXO7 stability and anti-TRAF2 co-immunoprecipitation. Additionally, the HindIII-XhoI fragments containing FBXO7 were ligated into pcDNA3.1/V5-His (Invitrogen) to generate Tyr52 and Cys52 FBXO7 cDNA in-frame fused to the V5-His for Western blot analysis of NF-κB signaling pathway protein TRAF2.

The VKOR coding sequence was PCR amplified from human liver cDNA (Clontech) using primers: VF1 (5′-CACCATGGGCAGCACCTGGGGGA-3′) and VR1 (5′-GCTCAGTGCCTCTTAGCCTT-3′) and TA-TOPO cloned into pcDNA3.1-V5/His (Invitrogen). The VKOR coding sequence was released by HindIII and NotI and then subcloned into the pcDNA3.1Hygro vector using the same restriction enzymes to give construct pVKORhygro (GenBank accession DM079693).

The complete coding sequences of human PRDX1-6 were amplified from HeLa cell cDNA by PCR and ligated into pcDNA3.1-V5-His (Invitrogen) for transient transfection. PRDX2 or PRDX4 cDNA was also ligated into pLenti4-puro for stable transfection. The catalytically inactive PRDX2(C51S) and PRDX4(C124S) mutants were generated using QuickChange Site-directed Mutagenesis Kit (Stratagene). PRDX2 and PRDX4 shRNA oligonucleotides were ligated into Teton-pLKO (Addgene #21915). HIF-1α and HIF-2α shRNAs were cloned into pLKO.1. The oligonucleotide sequences of shRNAs are shown in Table 1. Other constructs have been described previously [41 (link), 42 (link)]. The DNA sequences of all recombinant plasmids were confirmed by nucleotide sequence analysis.

Huh7.5 cells were kindly provided by Charles Rice (Rockefeller University, New York, NY) [15] (link). The expression vector pcDNA3.1/V5-His was from Invitrogen (Carlsbad, CA). The shRNAs targeting TRAIL, RIG-I shRNA targeted human RIG-I, and negative control shRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against human CD81 (5A6) and IRF-3, rabbit polyclonal anti-RIG-I antibody (H300), goat polyclonal anti-Noxa antibody (N-15), goat anti-mouse or goat anti rabbit IgG-HRP secondary antibody were from Santa Cruz Biotechnology. Monoclonal antibodies against β-actin and PUMA (10D4G7) were from Sigma. Rabbit polyclonal anti-ISG12a antibody (ab14695) was from ABCAM. Rabbit monoclonal anti-Cytochrome c antibody (136F3) and anti- PARP antibody (46D11) were from Cell Signaling Technology. Supersignal West Pico Chemiluminescent Substrate was from Pierce. 2′-O-me-anti-miR-942 (5′-CACAUGGCCAAAACAG AGAAGA-3′) and 2′-O-me-control-miR (5'-AAGGCAA GCUGACCCUGAAGU-3′) were from Takara. Mouse monoclonal anti-NS5A (HL1126) was a gift from Chen Liu (University of Florida, Gainesville, FL).

T cell transcription factor (TCF)/LEF response was measured using Cignal TCF/LEF reporter (luc) kit (SABiosciences). Activator protein 1 (AP1)/JUN pathway response was measured using a construct created by cloning an oligo containing seven copies of the AP1 binding element (TGACTAA) into a luciferase construct pGL4.10 (luc2) (Promega) at its KpnI and XhoI sites. pRL-TK was used for normalization (Promega). nuclear factor of activated T cells (NFAT) response was also measured using a luciferase reporter construct (plasmid number 10959; Addgene).
Plasmids modulating Wnt signaling pathway were used as comparator to show effect on aldosterone of activation or suppression of the canonical pathway. To activate the pathway, pcDNA3 δN47 β-catenin was used; and to suppress the pathway, pcDNA/Myc δN TCF4 (both from Addgene) were used. Where indicated, the vector control, pcDNA3.1-V5His (Invitrogen), was used. To measure luciferase (and renilla for normalization), a Dual-Glo luciferase assay system (Promega) was used according to the manufacturer's protocol. The selective porcine inhibitor, LGK-974 (1 μM; Selleck Chemicals) (21 (link)), and the selective c-Jun N-terminal kinase (JNK) inhibitor, JNK-IN-8 (1 μM, Selleck Chemicals) (22 (link)), were used to analyze the effect of blocking, respectively, all Wnt secretion or just the noncanonical Wnt signaling pathway.

For eukaryotic expression vector construction, the open reading frame of p66shc was amplified by PCR using the primers ePS with Hind III site and ePA with EcoR I site (Table 1), and sub-cloned into the plasmid expression vector pcDNA3.1/V5/His (Invitrogen), and the recombinant was designated pcDNA3.1/p66shc/V5/His.