Irf4.3e4

Antibodies against TCRγδ (GL3), TCRβ (H57-597), CD3 (17A2), Foxp3 (MF23), CD25 (PC61), PD1 (J43), Annexin V, IFN-γ (XMG1.2), NK1.1 (S17016D), CD27 (LG.3A10), IL-23R (12B2B64), IL-17A (TC11-18H10), CD45 (A20), CCR2 (SA203G11), CCR5 (HM-CCR5), CCR6 (29-2L17), CXCR6 (SA051D1), ICOS (C398.4A), CD62L (MEL-14), Ki-67 (SoIA15), CD44 (IM7), IRF4 (IRF4.3E4), and Blimp1 (5E7) were purchased from Biolegend. Antibodies against p-AKT (Ser473) (SDRNR), p-HIF-1α (Mgc3), and p-mTOR (Ser2448) (MRRBY) were purchased from Thermo Fisher. Before cell staining, TruStain FcX™ PLUS (anti-mouse CD16/32) antibodies (Biolegend) were used to block nonspecific binding. For intracellular staining of cytokines, the cells were stimulated with Cell Activation Cocktail (with Brefeldin A) from Biolegend. Six hours later, the cells were stained with antibodies against surface molecules and prepared according to the instructions of the Cyto-Fast™ Fix/Perm Buffer Set (Biolegend). For intracellular staining of transcription factors and signaling molecules, the cells were prepared with a Foxp3/Transcription Factor Buffer Set (Thermal Fisher).

Proteins extracted from cells or isolated by Co-IP were separated in 4–20% SurePAGE, Bis–Tris Gels (M00655; GeneScript) at 200 V for 30 min, and then transferred onto a nitrocellulose membrane at 150 mA for 2 h. Immunoblotted proteins on the membrane, labelled with specific antibodies were imaged by autoradiography. All primary and secondary antibodies were used according to the manufacturer’s instructions. The primary antibodies used were: IRF4 (3E4) (#646402; BioLegend); GR-HRP (sc-393232; Santa Cruz Biotechnology); β-actin (MAB8929; R&D systems); lamin A/C (E-1) (sc-376248; Santa Cruz Biotechnology).