(A) Co-immunoprecipitation of NOTCH2 and NOTCH2NL analyzed by immunoblot.
IP=Immunoprecipitation, N2NL=NOTCH2NL (B,C) Co-transfection of
NOTCH2-GAL4 and NOTCH2NL in the pGL3-UAS reporter assay in co-culture with U2OS
cells (B) or U2OS-JAG2 cells (C), n = 24 in 4 experiments. (D)
Immunoblot showing immunoprecipitated NOTCH2NL in the medium of mouse ESCs
ectopically expressing NOTCH2NL. (E) Effect of NOTCH2NL-conditioned medium in
NOTCH reporter assay, n = 7 in 2 experiments. One-way anova with
Tukey’s HSD (* p<10−4). (F–G)
NOTCH reporter assay using the NOTCH1 receptor in conjunction with NOTCH2NL.
(H–K) Assessment of common NOTCH2NL variants in reporter assay.
(H–I) n = 12 in 2 experiments. (J–K) n = 6 in 1
experiment, t-test with Holm-Bonferroni correction (* p<0.05,
** p<10−3), error bars indicate SD. Data
is analyzed using Two-way anova with Tukey’s HSD (*
p<10−4, **
p<10−8, ***
p<10−12). Error bars indicate SEM, unless otherwise
specified. See also Figure
S6.