Figure 5.

RFNG does not alter the steady-state levels of the NOTCH3 WT or C185R protein.A, representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells with and without doxycycline-inducible expression of RFNG. NOTCH3 and RFNG were detected by antibodies against human NOTCH3 extracellular domain (ECD) and human RFNG. TUBULIN was blotted with an antibody against human/mouse α-tubulin as a loading control. The white and black arrowheads indicate full-length NOTCH3 and the ECD, respectively. B, representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells treated with or without 0, 0.1, 0.5, or 1 μg/ml doxycycline. RFNG was detected by an antibody against HA, which tagged RFNG. The black arrowheads indicate N3ECDs. C, relative amounts of N3ECD WT and C185R with or without RFNG expression, as determined by Western blotting (B) (independent biological replicates N = 3). N3ECD band intensities were normalized to those observed without doxycycline. D, representative Western blot of cell lysates collected from cultured N3WT-RF-HeLa and N3C185R-RF-HeLa cells with or without RFNG expression and treated with puromycin for 0 or 16 h. The black arrowheads indicate N3ECDs. E, relative band intensities of N3ECD at 16 h normalized to those observed at 0 h, as determined by Western blotting in (D) (independent biological replicates N = 3). Data information: In C and E, data are presented as mean ± SD. n.s., not significant (C: Tukey’s post hoc test following one-way ANOVA; E: Tukey’s post hoc test following two-way ANOVA). HA, hemagglutinin; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.