Fig 2: A negative feedback loop from ERK to SOS facilitates ERK paradoxical activation by RAF inhibitors (A) A diagram of a structure-based dynamic model of the ERK pathway featuring negative feedback loops from ERK to RAF and SOS (see STAR Methods).(B and C) Time courses of the ERK activity responses to RAF inhibitors, which induce (B) or do not induce (C) RAF dimerization.(D and E) Steady-state ERK signaling responses to type I½ RAF and MEK inhibitors and their combinations are analyzed using Loewe isoboles. IC25 and IC50 lines correspond to the drug doses that achieve 25% or 50% ERK inhibition, respectively. In cells with a BRAFV600E mutation and WT RAS synergy between type I½ RAF and MEK inhibitors is observed at wider dose ranges for (D) low (~25 nM RAS-GTP of 750 nM total RAS) than (E) high WT RAS activities (~100 nM RAS-GTP of 750 nM total RAS).

Fig 3: Effects of epoxomicin and trehalose on ERK-1/2 and HSP70 chaperone protein activation in HD fibroblasts.(A) Western blot of p-ERK-1/2 expression with regard to total ERK and its corresponding densitometric analysis in control and HD fibroblasts. (B) Western blot of HSP70 expression and its corresponding densitometric analysis. Values are expressed as the mean ± SD, n = 4 patients. The data of each patient was obtained using 4 replicates. Statistical analysis was performed by one-way ANOVA with repeated measures followed by Bonferroni multiple comparison test: *p<0.05, ***p<0.001 vs Solvent; +++p<0.001 HD vs controls, ΔΔΔp<0.001 trehalose + epoxomicin vs epoxomicin. There is an interaction between epoxomicin effect and genotype in ERK activation (F = 71.13 with a p value = <0.0001). In HD, there is an interaction between the epoxomicin and trehalose effects in ERK activation (F = 12.67 with a p value = 0.0013).

Fig 4: Effect of various signaling components on ERK activation by GnRH. COS7 cells were co-transfected with plasmid-containing mouse GnRHR, together with each of the following plasmids: K721A-EGF receptor (Dn-EGFR); CD8-tagged ßARK (ß? scav); N-17 Ras (Dn-Ras); Csk-pRK5 (Csk); ß-arrestin2 (Arr); V54D-ß-arrestin2 (Dn-Arr); dynamin (Dyn); K44A-dynamin (Dn-Dyn); human FAK (FAK); and N-terminally truncated FAK (Dn-FAK). Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M; +) or left untreated (-). Phosphorylated ERK 1 and ERK2 were detected with anti-DP-ERK antibody (a-DP-ERK). The amounts of total ERK1 and ERK2 were detected with anti-ERK antibody (a-G-ERK). The results in the bar graph represent the percent activation of that obtained in the GnRH-a-stimulated cells that were co-transfected with GnRHR and vector control in each experiment. The results are averages of three experiments. Note: * p < 0.05.

Fig 5: Map3k1mPHD ES cells exhibit defective JNK and p38 activation following TGF-ß, EGF and nocodazole stimulationA–D WT and Map3k1mPHD ES cells were kept on low serum and stimulated with (A) TGF-ß (10 ng/ml), (B) EGF (100 ng/ml), (C) sorbitol (500 mM) or (D) nocodazole (0.5 µg/ml) for 10, 30 and 60 min or left unstimulated. Cells were lysed and analysed by IB using the indicated antibodies.Data information: Results are representative of three independent experiments.Source data are available online for this figure.