Figure 1.

Activation of NF-κB and NFAT in ROR1-CAR-T Cells after Antigen Stimulation

(A) Phenotype of CD4⁺ and CD8⁺ ROR1-CAR-T cells. Staining was performed with anti-EGFR mAb to detect the EGFRt transduction marker and AF647-labeled ROR1 protein to detect the CAR. (B) NF-κB and NFAT were detected by western blot in the nuclear and cytosolic fractions of ROR1-CAR-T cells. ROR1-CAR-T cells were either unstimulated or stimulated for 120 min with anti-CD3 and anti-CD28 mAbs, immobilized ROR1 protein, or PMA and ionomycin (P/I). Lamin A/C and α-tubulin served as loading controls for the nuclear and cytosolic fractions, respectively. (C) Nuclear enrichment of NF-κB and NFAT as quantified by densitometric analysis of western blots (n = 3 donors for CD4⁺ and n = 2 donors for CD8⁺ ROR1-CAR-T cells). Data were normalized to corresponding loading controls and presented as fold change ± SD relative to unstimulated T cells.