The transfections of miR-802 mimic, NC mimic, miR-802 inhibitor, NC inhibitor, si-Prkab1, and OE-Prkab1 into FL83B cells and murine primary hepatocytes were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The miR-802 mimic and its negative control (NC mimic), as well as the miR-802 inhibitor and its negative control (NC inhibitor) were purchased from GenePharma (Shanghai, China). The small interfering RNA targeting Prkab1 (si-Prkab1) was obtained from RiboBio (Guangzhou, China). All the sequences used in the transfections are shown in Table S5. The recombinant OE-Prkab1 plasmid was re-constructed as described in the following section.

Before transfection, 1×105 cells were plated in a 24-well format with 500 μl DMEM containing 10% FBS without antibiotics. The cells were 90% confluent at the time of transfection. Two μl of Lipofectamine 2000 reagent was added into 50 µl serum-free medium, which mixed with 0.8 µg of DNA and incubated for 20 min, and then the mixture was added into the cells. Cells incubated at 37 °C in CO2 incubator for 6 hours. Then the medium would be replaced with DMEM containing 10% FBS. The RNA or protein extraction was 24-48 hours after transfection 56.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.