Immune Cell Stimulation and Analysis

Functional grade anti-CD3 (145-2C11), anti-CD28 (37.51), anti-IL-4 (11B11), and anti-IFN-γ (H22) were purchased from BioXCell (West Lebanon, NH, USA). Fluorescently labeled anti-B220 (RA3-6B2), anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD25 (PC61), anti-NK1.1 (PK136), and anti-TCR-β (H57-597) were purchased from BioLegend (San Diego, CA, USA). Fluorescently labeled anti-phospho-c-Jun (Ser63) (KM-1) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-ATF4 (D4B8), anti-GRP78, anti-PERK (C33E10), anti-phospho-PERK (16F8), anti-eIF2a (D7D3), anti-phospho-eIF2a (Ser51) (D9G8), anti-phospho-p65 (Ser536) (93H1), anti-IκBα, anti-phospho-IκBα (Ser32), and anti-phospho-Erk1/2 (Thr202/Tyr204) (D13.14.4E) were provided by Cell Signaling Technologies (Danvers, MA, USA). FAT10, UBA6, and USE1 antibody were described previously [9 (link),10 (link),13 (link)]. The recombinant murine IL-1β (rmIL-1β), IL4, IL-6, IL-12p70, IL-23, and TGF-β1 were purchased from BioLegend (San Diego, CA, USA).