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. 2018 Dec 12;9:2944. doi: 10.3389/fimmu.2018.02944

Figure 2.

Figure 2

Abnormal NF-κB activation induced by H234LΔ235-8 CARD11 in vitro. (A) JPM50.6 T cells were transfected with 5 μg of either empty vector (EV), WT, E134G (GOF mutant), or H234LΔ235-8 mutant CARD11-FLAG plasmids alone (top panel) or in the presence of 5 μg WT CARD11 plasmid (bottom panel) as previously described (14). After 24 h incubation in complete RPMI, transfected cells were stimulated with 1 μg/ml of anti-CD3 and anti-CD28 Abs or left unstimulated for an additional 24 h. GFP expression (reflecting relative NF-κB activity) was subsequently measured by flow cytometry; %GFP cells are labeled in each histogram. (B) Quantification of NF-κB driven GFP reporter expression in transfected JPM50.6 cells as described in (A). Data are mean ± SEM for mean fluorescence intensity of GFP+ cells for 3 separate transfection experiments. Asterisks denote statistically significant differences (2-way ANOVA) between the stimulated groups indicated. (C) Immunoblots confirming the comparable expression of WT and mutant CARD1-FLAG proteins in cells from (A) at 24 h post-transfection. β-actin served as a loading control. Data are representative of 3 separate experiments. (D) WT Jurkat T cells were transfected with WT or mutant CARD11-FLAG plasmids as in (A). After 24 h, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 20 min and lysed. Immunoprecipitations (IPs) using anti-BCL10 Ab were performed as previously described (12). BCL10 IPs and input lysates were separated by SDS-PAGE and immunoblotted Abs against CARD11, BCL10, and MALT1. (E) JPM50.6 cells were transfected with WT CARD11-GFP –/+ WT or mutant CARD11-FLAG plasmids as in (A). After 24 h, cells were stimulated with PMA for 20 min and lysed. FLAG IPs and input lysates were immunoblotted for total CARD11 protein; arrows indicate GFP- vs. FLAG-tagged CARD11. Actin served as a loading control for input lysates. IPs in (D,E) are representative of 2 separate experiments each. (F) PBMC from 2 healthy controls (HC) and patients III.1 and IV.1 were stimulated with PMA (20 ng/ml) plus monensin (2 μM) for 20 min. Cells were stained with FITC-conjugated mouse anti-human CD4 mAb, fixed in 1.5% paraformaldehyde and permeabilized in ice cold methanol before staining with AlexaFluor647-conjugated mouse anti-human phospho-p65 (Ser529) or AlexaFluor647-conjugated mouse anti-human IκBα. NF-κB activation was assessed in gated CD4+ T cells by flow cytometry; numbers in each histogram denote % of phospho-p65+ (left) or IκBαhi cells (right). Data are representative of 2 separate experiments.