Nucleofector kits for mouse t cells

The Il21 promoter and conserved noncoding sequences were PCR amplified using listed primers and cloned into pGL3 luciferase plasmid. Plasmids containing Batf mutated sites were constructed using QuikChange XL Site-Directed Mutagenesis Kit (Agilent). The Batf-expression vector was previously described (Jabeen et al., 2013 (link)). For T cell transfection experiments, naive CD4⁺ T cells were activated with 2 ug/ml anti-CD3 and 0.5 ug/ml anti-CD28 with or without 20 ng/ml IL-6. After 72 h, cells were transfected with the luciferase plasmids and control plasmid using Nucleofector Kits for Mouse T Cells (Lonza). Cells were rested overnight, restimulated with PMA and ionomycin for 5 h, and followed by assessment of luciferase expression with the dual luciferase system (Promega).

Small interfering RNA (siRNA) kits (Origene) were used to knock down the expression of hnRNP A1 and hnRNP L in mouse primary CD4⁺ T cells. The nucleofector kits for mouse T cells (Lonza) and Amaxa nucleofector were used to introduce siRNAs into murine CD4⁺ T cells. To measure the knockdown efficiency, both qPCR analysis and Western blot analysis were performed in triplicate.