RLTPR selectively activates the NF-κB pathway in a TCR-dependent manner. (A) Volcano plot analysis of differentially expressed RNA transcripts in unstimulated RLTPR (p.Q575E) vs RLTPR WT Jurkat cells. Dotted line indicates adjusted P value = .01. (B) Volcano plots of differentially expressed RNA transcripts in PMA/ionomycin-stimulated RLTPR (p.Q575E) vs RLTPR WT Jurkat cells. Genes that are above the dotted line represent the genes having statistical significance with adjusted P value < .01. Red and blue dots represent upregulated and downregulated genes, respectively. (C) Expression of RLTPR-dependent genes (genes indicated in red in panel B) as a function of RLTPR mutation status and stimulation. Data represent the ratio of normalized mRNA transcript counts in each subgroup compared with unstimulated RLTPR WT Jurkat cells. P value was determined by 2-sided paired ratio t test. n.s., not significant; ****P < .0001. (D) Gene set variation analysis of RNA-seq data comparing signaling pathways differentially activated in PMA-ionomycin activated RLTPR (p.Q575E) Jurkat cells compared with PMA-ionomycin activated WT controls. (E) Identification of transcription factor binding motifs that are selectively upregulated by the RLTPR mutation. OPOSSUM analysis was performed on the 96 RLTPR mutant-dependent gene transcripts; that is, the genes indicated in red in panel B. The graph shows the 4 highest ranked transcription factors, ranked by lowest adjusted P values. (F) Immunoprecipitation assay assessing the interactions between FLAG-tagged RLTPR and CARMA1 according to mutation status and stimulation status. Lysates from Jurkat cells are subjected to co-immunoprecipitation using anti-Flag beads, and blotted with anti-CARMA1 antibody. β-actin was used as the loading control. Immunoblotting for FLAG, RLTPR, and CARMA1 were used to confirm equivalent expression of the FLAG-tagged RLTPR and CARMA1 in each input sample.