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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 1. ARAF S214 mutants signal as RAS-independent dimers but do not result in feedback inhibition of cellular RAS-GTP. See also Figure S1.

Figure 1.

(A) K-Raslox MEF cells transduced with retrovirus carrying doxycycline-inducible WT ARAF or the indicated mutants were grown in medium without or with 1 μM 4-OHT for a week to generate isogenic cells expressing or lacking K-RAS. Cells were then treated with 100 ng/ml doxycycline for 24 hours and subjected to western blot.

(B) K-Raslox MEF cells lacking K-RAS expression were transfected with indicated constructs and subjected to western blot.

(C and D) NIH3T3 cells expressing doxycycline-inducible RAF variants were treated with doxycycline for 48 hr. Signaling activity and RAS-GTP levels were then examined by western blot.

(E) NIH3T3 cells expressing doxycycline-inducible RAF variants were treated with 100 ng/ml doxycycline for 24 hours. Relative expression levels of ERK target genes were examined by qRT-PCR. Error bars, mean±SD triplicate of experiments.