Double luciferase reporter assay kit

pLV-β-catenin deltaN90 was a gift from Bob Weinberg (Addgene plasmid # 36985) [28 (link)]. M50 Super 8 × TOPFlash (Addgene plasmid # 12456) and M51 Super 8 × FOPFlash (Addgene plasmid # 12457) were gifts from Randall Moon [29 (link)]. ME180 cells were transfected with plasmid DNA using X-tremeGENE™ HP DNA Transfection Reagent (Roche, 06366236001) for 24 h. Cells were lysed and subjected to luciferase reporter assay by using a Double-Luciferase Reporter Assay Kit (TransGen Biotech, Beijing, China) and was normalized to pSV40-Renilla luciferase activity.

The proximal promoter of zebrafish il22 (−2719 to + 506 bp) was amplified with the primers listed in Supplemental Table 2. The il22 promoter fragment was cloned into linearized (by using HindIII) pGL3-Basic luciferase reporter plasmid. The pGL3 (control or containing il22 promotor) (20 ng/uL) and pRL-CMV (2 ng/uL) plasmids were microinjected into the embryos at one or two-cell stage based on a method described previously.^{46 (link)} Subsequently, embryos were incubated with control buffer or 1,25(OH)₂D₃ (10 nM) for 24 h. The relative luciferase activity was assessed by using a Double-Luciferase Reporter Assay Kit (Transgene Biotech, FR201, China) and normalized to the Renilla luciferase activity (pRL-CMV).

Rabbit monoclonal anti-IRF3, GAPDH, eGFP-Tag, HA-Tag and HRP-conjugated goat anti-rabbit IgG antibodies, and mouse monoclonal anti-Myc-Tag Flag-Tag antibody were purchased from Cell Signaling Technology (USA). The JetPRIME kit was purchased from Polyplus Transfection (France) and Double-Luciferase Reporter Assay Kit was purchased from TransGen (China). HRP-conjugated goat anti-mouse-IgG antibody, protease inhibitor, and phosphatase inhibitor were provided from CWBIO (China). The Pierce Crosslink Magnetic IP/Co-IP Kit was purchased from Thermo Scientific (USA). Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) were purchased from Abcam (USA). TRIM21 Polyclonal antibody was purchased from Proteintech (USA). MG132, chloroquine diphosphate (CQ), 3-methyladenine (3-MA) and Z-VAD-FMK were purchased from MedChemExpress (MCE) (USA).

The cloned FfMYB15 sequence was inserted into the pBD plasmid (StuI) provided by Bing Deng [58 (link)]. The recombinant pBD-FfMYB15, pBD (negative control), and pBD-VP16 (positive control) plasmid were used as effectors. The modified pGreenII0800-LUC plasmid was used as reporter, which contains Ren and Luc. Ren was driven by CaMV35S promoter and Luc was driven by the minimal TATA box of CaMV35S promoter with 5 × GAL4 binding elements. Reporters and effectors were transferred into EHA105 and transferred to the infiltration buffer to adjust the OD600 to 0.8. Effectors and reporters were mixed in the ratio of 9:1 and injected into uniformly sized, well-growing tobacco leaves [58 (link)]. Tobaccos were cultured in the dark for 12 h in order to resume leaf growth, and then transferred to an environment representing normal culture conditions. After 48–72 h, the ratio of Luc/Ren in tobacco leaves was detected by Double-Luciferase Reporter Assay Kit (TransGen Biotech, Beijing, China). Six independent replicates were assessed.

The wild-type sequence of circWDR37 was subcloned downstream of the Renilla luciferase reporter gene of the pSI-check2 expression vector (Hanbio Biotechnology, Co., Ltd., Shanghai, China) named as WT-WDR37. The sequence of circWDR37 with 4 mutation sites in positions 76, 78, 80, and 82 was synthesized and subcloned into the downstream of pSI-check2 named as (M1-WDR37). The sequence of circWDR37 with 3 mutation sites in positions 108, 110, and 113 was synthesized and subcloned into the same site as WT-WDR37 in pSI-check2 (M2-WDR37). Double-stranded nucleotides were designed to mimic endogenous mature hsa-miR-328-3p (miR-328, miRBase ID: MIMAT0000752) and hsa-miR-138-5p (miR-138, miRBase ID: MIMAT0000844) synthesized by GenePhama Co., Ltd. (Shanghai, China). A scrambled RNA was used as a negative control (NC). WT-WDR37/M1-WDR37/M2-WDR37 mixed with miR-328/miR-138/NC was cotransfected into HEK-293 cells. Luciferase activities were measured with a double-luciferase reporter assay kit (TransGen Biotech Co., Ltd, Beijing, China) on a luminometer (GloMax 20/20, Promega, Co., Ltd, USA) after one-day culture.