(A and B) HA-tagged (A), full length, or CARD12 WT and CARD2 (B) mutants as indicated were transfected in HEK293T cells stably expressing IFN-β promoter driven firefly luciferase gene and luciferase activity (RLU) was measured 48 hr later. Protein expression levels were detected by western blotting using an HA-HRP antibody. β-actin was used as a loading control.
(C) Graph depicts arbitrary units of de-repression of CARD12 for each mutant calculated by the ratio of interferon induction (RLU) of full-length (FL) RIG-I to CARD12 RIG-I normalized to respective quantified band intensity ratios of HA to β-actin.
(D) Five-fold serially diluted empty plasmid, full-length or CARD12 WT or mutants were transfected in HEK293T cells stably expressing IFN-β promoter driven firefly luciferase gene and luciferase activity (RLU) was measured 48 hr later. Protein expression levels were detected by western blotting using an HA-HRP antibody. β-actin was used as a loading control. Dashed line indicates background luciferase expression and dotted line indicates the threshold of IFN-β induction by WT RIG-I. Data represents mean of three to four independent experiments with error bars showing standard deviation. Statistically significant differences are indicated with a p value.