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. 2023 Feb 15;9(7):eadd7969. doi: 10.1126/sciadv.add7969

Fig. 1. Trans-acting MEK phosphorylates KSR1 C809Y.

Fig. 1.

(A) KSR1 C809Y incorporates phosphorylated ERK. HEK293T cells were transfected with Flag-tagged wild-type (wt) or C809Y KSR1 (1.5 μg) and stimulated with EGF (50 ng/ml, 5 min) where indicated (+) after 18 hours of starvation (−). KSR1-associated proteins were determined by coimmunoprecipitation upon anti-Flag immunoprecipitation (IP: Flag) and subsequent Western blotting. TL, total lysate. (B) C809Y binds to phosphorylated ERK in live cells. Ectopic Flag-tagged C809Y interaction with endogenous ERK, determined by PLA in HeLa cells after starvation, in starved (st) or EGF-treated cells. Scale bar, 10 μm. (C) C809Y can homodimerize. HEK293T cells were transfected with the indicated Glu- and Flag-tagged KSR1 constructs and EGF-stimulated where indicated (+). Immunoprecipitations performed with a specific antibody (IP) or with preimmune serum (PI). un, untransfected cells. (D) KSR1 double mutant fails to bind phosphorylated ERK. Coimmunoprecipitation assay in HEK293T cells transfected with the indicated Flag-tagged KSR1 constructs, in starved cells (−) or upon EGF stimulation where indicated (+). (E) KSR1 double mutant fails to bind phosphorylated ERK in vivo. HeLa cells were transfected with the indicated KSR1 constructs (2 μg). PLA as in (B), in EGF-stimulated cells. Scale bar, 10 μm. (A and D) Figures show signal intensity relative to the levels found in untreated cells. All the results shown are representative of three to five independent experiments.