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

Figure 7.

Figure 7

RFNG does not affect JAG1-mediated transendocytosis of NOTCH3 WT and C185R.A, schematic representation of the immunocytochemistry workflow used to separately detect NOTCH3 present in the cell membrane and in JAG1-3T3 cells. After N3WT/C185R-RF-HeLa cells transfected with NOTCH2 siRNA were cocultured with JAG1-3T3 cells, the fixed cells were stained with two different anti-NOTCH3ECD (N3ECD) antibodies (clones 1G5 and BAF1559) before and after cell permeabilization, respectively. The scale bars indicate 20 μm. B, representative confocal images of cocultured N3WT/C185R-RF-HeLa and JAG1-3T3 cells, as depicted in (A). The white arrowheads indicate NOTCH3 particles detected by antibodies against N3ECD (clones 1G5 and BAF1559), indicating the presence of NOTCH3 on the cell membrane. The scale bars indicate 20 μm. C, the ratio of NOTCH3 present on the cell membrane against the total NOTCH3 in JAG1-3T3 cells. Box plots: 10 to 90 percentile. (independent biological replicates: N = 3; total cell numbers, WT RFNG-: n = 86, WT RFNG+: n = 86, C185R RFNG-: n = 93, C185R RFNG+: n = 81). ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (Tukey’s post hoc tests following two-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.