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. 2024 Sep 19;300(10):107787. doi: 10.1016/j.jbc.2024.107787

Figure 8.

Figure 8

RFNG impairs JAG1-stimulated signaling activity of NOTCH3 R141C and C185R more effectively than WT.A, a workflow for evaluating the JAG1-mediated signaling activity of NOTCH3 WT and C185R, with NOTCH2 knockdown. N3WT/C185R-RF-HeLa cells were transfected with NOTCH2 siRNA on day 0. The cells were transfected with the Notch reporter plasmid, and doxycycline was added or not added on day 1. Subsequently, MIG-3T3 or JAG1-3T3 cells were seeded on day 2, and total cell lysates were collected on day 3. B, Notch reporter assay with the collected lysates from cocultures of N3WT/C185R-RF-HeLa and JAG1-3T3 or MIG-3T3 cells. Values were calculated relative to that observed upon coculture with MIG-3T3 cells (independent biological replicates N = 4). C, fold change in JAG1-mediated activities of NOTCH3 WT and C185R with RFNG coexpression against those without RFNG (independent biological replicates N = 4). D, fold change in JAG1-mediated activities of NOTCH3 WT and R141C with RFNG coexpression against those without RFNG (independent biological replicates N = 5). E, fold change in JAG1-mediated activities of NOTCH3 WT and R90C with RFNG coexpression against those without RFNG (independent biological replicates N = 5). Data information: In B-G, data are presented as mean ± SD. n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (B, D, and F: Tukey’s post hoc test following three-way ANOVA; C, E, and G: Unpaired one-tailed Student’s t test). JAG1, Jagged 1; RFNG, Radical fringe.