Fig. 4: NTF releases cysteines in NOTCH3 and makes them accessible for thiol-labeling.

Following the addition of NTF to Fc-tagged NOTCH3 containing the first 3 EGF-like repeats, we observed increased maleimide signal with increased lengths of incubation with NTF until 24 h, after which signal decreased (a). Detection of NTF using an antibody specific for the cleaved fragment of NOTCH3, UMI-D, revealed a complex at a size larger than the NOTCH3 alone size (b; green). Purified NOTCH3 was identified by probing for the Fc tag (b; red). We observed increased maleimide signal with the addition of NTF to all WT and mutant samples, while the addition of NTF to Fc protein alone did not increase signal (c, upper panel). A parallel set of samples was transferred to nitrocellulose using the iBlot 2 system and protein loading amounts were verified by probing for Fc (c, lower panel). Quantification of maleimide signal in samples with and without NTF demonstrated 5.13-fold enhancement in WT protein and 24.37-fold, 28.55-fold, 29.26-fold, and 20.44-fold enhancement in mutant proteins R90C, C49Y, R75P, and R141C, respectively (d). The addition of NTF to Fc resulted in 20% signal compared to without NTF (d). Effects of NTF on purified WT NOTCH3 were compared to those from a form of NTF with all cysteines mutated to serine (6S). The addition of NTF to NOTCH3 resulted in an increased maleimide signal, while no increase was observed with the addition of 6S to NTF (e). Similarly, no increase was observed with the addition of two concentrations (0.1 and 0.5 mM) of two other sulfhydryl-containing compounds, glutathione and homocysteine (f). NTF was then added to purified Fc-tagged NOTCH ectodomain fragments for increasing lengths of time. Cysteines were then capped with 10 μM IRDye 800CW Maleimide (g, left panel). Total protein content was determined by SimplyBlue Safestain (ThermoFisher) (g, right panel). Quantification of maleimide signal normalized to total protein amount revealed increasing maleimide signal that peaks at 1 h for all mutants and decreases with longer incubation times (h). WT protein signal peaks around 24 h. Experiments were repeated three times using distinct samples with similar results, and (h) plots the mean of three replicates. Unprocessed western blots can be found in Fig. S10.