Table 1. The interaction of type 2 RBP-J mutants with the ANK repeats of IC.

		DNA	GST pull-down assays				Transcriptional activity
		binding	ANK	RAM	CID	KyoT2	RAMIC	IC	ANK–VP16
	wild-type RBP-J	100	100	100	100	100	100	100	100
5a	KR185GS	104	109	114	92	108	71	56	83
6	S194A	92	92	103	90	102	41	39	47
7a	KK196GS	102	135	101	90	99	49	53	67
5a+6	KR185GS+S194A	92	146	110	95	107	18	27	35
5a+7a	KR185GS+KK196GS	106	116	108	81	98	19	38	73
6+7a	S194A+KK196GS	104	95	107	80	97	23	34	55
26	InsRsa1265	96	131	95	70	88	11	0	0

Relative transcriptional activities induced by Notch IC and ANK–VP16 (and RAMIC) of RBP-J mutants compared to wild-type RBP-J were evaluated by the same approach as described in Figure 1A (1 µg of pEFBOSneo IC or 0.25 µg of pEFBOSneo ANK–VP16 was reacted as an activator). Relative affinities of RBP-J mutants for GST fusion proteins were also evaluated in the GST pull-down assays as described in Figure 2D. To detect relative DNA-binding properties, the same amounts of in vitro-translated type 2 RBP-J mutants were reacted with the ³²P-labeled O54 probes and evaluated by EMSA in a linear range (16). All activities are relative to wild-type RBP-J.