In vitro PRMT5 methyltransferase assay.

Methylation activity in nuclear extracts derived from indicated human T cells was measured using Epigenase PRMT5 Methyltransferase (Type II-Specific) Activity/Inhibition Assay Kit (EpiGentek) according to the manufacturer’s protocol. Standard curves were generated using Methylated H4-Arg3 standard provided by the manufacturer, with a linear detection range between 0.1 and 2 ng of methylated product. Reactions contained 20 μg of the indicated nuclear extracts (prepared with Nuclear Extraction Kit, OP-0002 from EpiGentek) and were incubated for 120 minutes at 37°C. Absorbance (450 nm with a reference wavelength of 655 nm) was measured with a Tecan Infinite M1000Pro microplate reader (Thermo Fisher Scientific). Each experiment included at least 3 independent replicate measurements per sample, and P values for each sample comparison were calculated using a 2-tailed unpaired t test.