Fig. 2.

Hyperactivation of TCR signaling in FAM49B-deficient Jurkat T cells. (A) Schematic of validating screen candidate genes by transducing Cas9⁺ Jurkat cells with lentivirus-expressing sgRNA at an MOI of 0.5. (B) FACS analysis of CD69 levels in Jurkat cells expressing sgRNAs targeting NTC, c-Cbl, or FAM49B 13 h after anti-TCR C305 stimulation. Data are representative of three independent experiments. ***P < 0.0001, **P < 0.001 (Student’s t test). (C) FACS analysis of pERK phosphorylation in the sample described in B. Histogram shows pERK MFI from 0 to 60 min upon anti-TCR stimulation. (D) Schematic of reconstitution J.FAM49B cells and J.NTC cells with empty vector (GFP) or FAM49B vector, and FACS analysis of pERK level upon anti-TCR stimulation. (E) The FAM49B-suffcient and -deficient cells were rested and stimulated with anti-TCR antibody for times indicated. Phosphorylation of Lck, Zap70, LAT, SLP-76, PLCγ, and ERK were assessed by immunoblotting.