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. Author manuscript; available in PMC: 2023 Jul 7.
Published in final edited form as: Mol Cell. 2022 May 24;82(13):2443–2457.e7. doi: 10.1016/j.molcel.2022.04.034

Figure 3. ARAF increases RAS-GTP by competing with RASGAP NF1 for binding to RAS. See also Figure S3.

Figure 3.

(A) GTP hydrolysis reactions were assembled with purified 0.5 μg (1 μM) N-, H-, or K-RAS and NF1-333 (50 ng/50 nM for N- or H-RAS, 500 ng/500 nM for KRAS) in the presence/absence of RAF proteins (60 nM to 1.8 μM). Reactions were incubated for the indicated time points and luminescence was recorded. Bars, mean±SD biological triplicate of experiments.

(B) SKBR3 cells transfected with indicated constructs were collected 24 hours after transfection. Endogenous RAS was immunoprecipitated.

(C) 293FT cells co-transfected with FLAG tagged N-, H- or K-RAS, NF1 and different amounts of RAF constructs were collected 24 hours after transfection. NF1 was co-immunoprecipitated with RAS and the NF1-RAS binding was quantified and normalized to basal levels.

(D and E) Cas9 expressing SKBR3 cells were transfected with sgRNAs targeting NF1. After 48 hours, cells were transfected with indicated siRNAs (D), or constructs (E).