Prl tk renilla

A pGMFOXO-Lu (Genomeditech, Shanghai, China), a pRL-TK Renilla (Beyotime, Nan-tong, China) and AMPK siRNA or FOXO3a siRNA were transfected into RAW 264.7 cells using Lipofectamine 2000™ reagent (Invitrogen, CA) [66 (link)]. Then cells, lysed by Promega passive lysis buffer, were assayed by using Promega dual luciferase (Firefly luciferase/Renilla luciferase) kit. Luciferase intensity detected with a Luminoskan Ascent (Thermo Fisher Scientific Inc. Finland).

IPEC-J2 cells (4 × 10⁴ cells/mL suspension) were plated in a 96-well plate for 24 h and transfected with 100 ng reporter plasmid TOP or FOP (Beyotime Biotechnology, Shanghai, China) and 10 ng pRL-TK/Renilla (Beyotime Biotechnology, Shanghai, China) for each well by Lipofectamine 2000 (Invitrogen, MA, USA). For NEDD4L and DVL2 evaluation, the 100 ng of additional plasmid (pcDNA-NEDD4L and pcDNA-DVL2) was co-transfected in each well when indicated. For miRNA evaluation, the mimics and mimics-NC were additionally transfected into corresponding wells. The luciferase intensities were observed by the commercial Dual-Glo Luciferase Assay Kits (Promega, WI, USA) 24 h later. The intensities of TOPFlash and FOPFlash were normalized to Renilla.

THP‐1 cells were transfected with the plasmids pRL‐TK Renilla (Beyotime) and pNF‐κB‐TA‐luc (Beyotime) using Lipofectamine 3000 (Invitrogen). Forty‐eight hours later, lysis buffer was used to lyse the cells, which were then centrifuged at 12,000 × g for 5 min. To collect the supernatant, the cell lysates were first mixed with 100 μL of firefly luciferase detection reagent (Beyotime) and measured for relative light units (RLUs), and then with 100 μL of Renilla luciferase assay solution, and the RLU was measured again. The ratio of the RLU of firefly luciferase to the RLU of Renilla luciferase is shown as relative luciferase activity.