Ab205016

Cells were cultured in 5% CO_2, 37°C. CD8 T cells were purified from mouse lymph nodes using an easySep CD8 kit (Stemcell Technologies) and activated on plate-bound anti-CD3 (clone 145–2C11; Biolegend) and anti-CD28 (clone 37.51; Biolegend) antibodies in RPMI medium (Gibco/Thermofisher) with 20 ng ml⁻¹ interleukin 2 (Novartis), 10% fetal calf serum (FCS) (Gibco/Thermofisher), 50 mM 2-mercaptoethanol (Sigma) and PenStrep (Thermofisher) for 5 days. Hela cells were grown in DMEM (Gibco/Thermofisher) with 10% FCS and 5 U ml⁻¹ penicillin-streptomycin (ThermoFisher). PCIF1-directed siRNA smartpool or scrambled siRNA (Dharmacon) was transfected using a Neon transfection system (Thermofisher) using 1005 V × 35 ms × 2. CAPAM knockdown was confirmed by western blotting with anti-PCIF1 (ab205016; Abcam) and anti-actin (ab3280; Abcam) antibodies. RNA was prepared from cells as per the tissue samples, with the exception that cells were directly lysed in Tri-Reagent rather than frozen and ground.