Fig 2: CTSV mediates GATA3 degradation through Akt and GSK-3ß signalling. a, b Western blotting analysis of pThr 308 Akt, pSer473 Akt, Akt, pSer9 GSK-3ß, GSK-3ß pSer308 GATA3 and GATA3 expression in MCF-7 and ZR-75-1 shCTSV cell line models. Restoration of CTSV expression in sh1 cells was enabled by the generation and transfection of an shRNA-resistant rescue construct (rCTSV). c, d Western blotting analysis of GATA3 expression following transfection of STYX (Flag-tagged) or pCMV empty vector (EV) into MCF-7 or ZR-75-1 cells. e, f Western blotting analysis of GATA3 expression in MCF-7 and ZR-75-1 cells following treatment with Buparlisib (250 µM, 6 h). GAPDH was used as an internal loading control for all Western blotting analysis

Fig 3: Cancer-associated mutations in switch III of N-ras do not alter its biochemical properties and Gal-1 complexation.(A) Gal-1-complexation FRET data of indicated N-ras mutants and parent construct transiently expressed in BHK cells. (B) RBD-recruitment FRET analysis of indicated N-ras mutant and parent constructs transiently expressed in BHK cells with (+) or without (-) 5 µM compactin treatment for 48 hr. Compactin treatment relocalized N-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (A, B) Numbers in bars give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant N-ras. +EGF denotes stimulation with 100 ng/ml EGF; -EGF serum starved cells; +GAP incubation with GAP domain of NF1, to assay for GAP-sensitivity. The graphs represent the averages of active N-ras mutants normalized to wt N-ras + EGF-stimulation from two independent experiments. Blue vertical line annotates the activity of wt N-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCit-N-ras-wt, mCit-N-ras-T50I, mCit-N-ras-E49K, or mCit-N-ras-C51Y. Cells were serum starved and collected without EGF stimulation (-EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the ß-actin row. (E) Validation of comparable protein expression levels of HA-tagged N-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI: http://dx.doi.org/10.7554/eLife.08905.013

Fig 4: K-ras with mutation R164Q has unaltered Gal-1-complexation and biochemical properties.(A) Gal-1-complexation FRET analysis of K-ras mutants and its parent construct transiently expressed in BHK cells. (B) RBD-recruitment FRET analysis of K-ras mutants and its parent construct transiently expressed in BHK cells with (+) or without (-) 5 µM compactin treatment for 48 hr. Compactin treatment relocalized K-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (A, B) Numbers in bars give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant, ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant K-ras. +EGF denotes stimulation with 100 ng/ml EGF. -EGF serum-starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active K-ras mutants normalized to wt K-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt K-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant, ***p < 0.001). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCFP-K-ras-wt, mCFP-K-ras-V152G, or mCFP-K-ras-R164Q. Cells were serum starved and collected without EGF stimulation (-EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the ß-actin row. (E) Validation of comparable protein expression levels of HA-tagged K-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI: http://dx.doi.org/10.7554/eLife.08905.015

Fig 5: The expression levels of Akt, p-Akt (Ser473), p-Akt (Thr308), FAK, and p-FAK (Tyr397) in WJMSCs monolayer/ microcarrier culture as determined by a Jess™ Simple Western automated nano-immunoassay system. (A) Representative image of Akt, p-Akt (Ser473), p-Akt (Thr308), FAK, and p-FAK (Tyr397) protein levels from Jess simple western blotting. (B) Specific activity of p-Akt (Ser473), p-Akt (Thr308), and p-FAK (Tyr397) to determine the phosphorylation of Akt and FAK in WJMSCs monolayer and microcarrier culture. p < 0.05 *.