Table 2.

Enhanced site 1a interactions do not fully compensate for the loss of site 1b binding interactions through the NTD

		Wild-type IL-3			IL-3 K116W
Cell	Receptor subunits	n	KD (nM)	p	n	KD (nM)	p	P
CTLL-2	IL3Rα + βc	2	0.34 ± 0.03	—	2	0.14 ± 0.01	—	—
TF-1	Endogenous	4	2.06 ± 0.35	—	3	0.35 ± 0.12	—	0.006
TF-1Hi	Overexpressed IL3Rα	7	0.14 ± 0.02	<0.0001	6	0.065 ± 0.006	0.003	0.004
COS	IL3Rα + βc	15	0.48 ± 0.05	—	6	0.43 ± 0.09	—	0.98
COS	IL3Rα SP2 + βc	10	7.61 ± 0.97	<0.0001	6	2.98 ± 0.87	0.011	0.004
COS	IL3Rα C76A,C195A + βc	8	1.10 ± 0.31	0.009	6	0.94 ± 0.26	0.11	0.69
COS	IL3Rα D197L + βc	6	4.37 ± 0.85	<0.0001	4	1.60 ± 0.09	<0.0001	0.02
COS	IL3Rα	8	140.6 ± 16.9	—	6	17.8 ± 5.8	—	<0.0001
COS	IL3Rα SP2	3	N.B.	—	6	N.B.	—	—
COS	IL3Rα C76A,C195A		n.d.		4	N.B.	—	—
COS	IL3Rα D197L		n.d.		4	N.B.	—	—

Binding of wild-type IL-3 or IL-3 K116W to cells expressing the indicated IL-3 receptor subunits was measured in saturation binding assays using radioiodinated cytokine^{60, 66}. The CTLL-2 cell line expresses full-length IL3Rα and βc was used as a basis for functional studies of IL3Rα mutants. The human TF-1 cell line or its derivative, TF-1 Hi that expresses high levels of IL3Rα as a result of being transduced with a lentivirus encoding human IL3Rα, were used as a model of human AML. COS cells were transfected with plasmids encoding wild-type, D197L or C76A,C195A forms of full-length IL3Rα or IL3Rα SP2, alone or together with βc. Errors represent SEM from the indicated number (n) of experiments or SD for n < 3. Statistical significance of differences in binding between TF-1 and TF-1Hi or wild-type IL3Rα and the mutant forms of IL3Rα (p) or between IL-3 and IL-3 K116W (P) was determined using a two-tailed unpaired t test. Related to Figs. 3, 4, 5, 6, Supplementary Figs. 4, 6, 8 and Table 3

n.d. not done, N.B. no binding