S805935

HEK293E cells were seeded on a 12-well plate. After 24 h, 500 ng of renilla (Switchgear Genomics S805935 MAPK13 3′UTR or MAPK13 m⁶A site mutant constructs) and 100 ng of cypridina (Switchgear Genomics SN0322S) luciferase constructs were cotransfected into cells using FuGENE HD (Promega). About 48 h after transfection, luciferase activity was measured using LightSwitch Renilla Luciferase Assay reagent (Switchgear Genomics) and Pierce Cypridina Luciferase Glow Assay kit (Pierce) on Victor Nivo plate reader (PerkinElmer) according to the manufacturer’s protocols. The activity of renilla luciferase was normalized by cypridina luciferase activity.

The point mutation of m⁶A modification site (A1212 to T1212) of human MAPK13 3′UTR luciferase reporter (Switchgear Genomics S805935) was generated using a QuickChange site-directed mutagenesis kit according to the manufacturer’s protocol using Pfu Ultra polymerase (Agilent Technologies). Mutagenesis primers are MAPK13-3UTR-GGACC-mut-fw (5′-CACTGCCCAAGGTCCAGTATTTGTC-3′) and MAPK13-3UTR-GGACC-mut_rv (5′-GACAAATACTGGACCTTGGGCAGTG-3′).