Transfection of 293T cells with CN mutants allows luciferase expression in the presence of CN inhibitors. 293T cells were transfected with a CN mutant, a Firefly luciferase reporter gene driven by an NFAT response element, and an internal control Renilla luciferase plasmid. Cells were activated with PMA and ionomycin, and the expression of luciferase was assessed. Resistance was determined by comparing luciferase expression upon stimulation in the presence of CN inhibitors to luciferase expression upon stimulation in the absence of CN inhibitors. Mean and SEM of 3 experiments shown. (A) Screening of CNa mutants identifies 6 that confer resistance to 10 ng/mL FK506 (CNa2, 4, 9, 12, 15, and 17) and 5 that confer resistance to 200 ng/mL CsA (CNa18-CNa22). (B) Screening of CNb mutants identifies 5 that confer resistance to 10 ng/mL FK506 (CNb12, 21, 22, 23, and 30) and 5 that confer resistance to 200 ng/mL CsA (CNb21, 22, 23, 26, and 30). Of the CNb mutants identified, 4 confer resistance to both FK506 and CsA (21, 22, 23, and 30).