MOESM1 of Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling
Creators: name: Mehraj-U-Din Lone
name: Miyan, Javed
name: Asif, Mohammad
name: Showkat Malik
name: Dubey, Parul
name: Singh, Varsha
name: Singh, Kavita
name: Mitra, Kalyan
name: Pandey, Deepali
name: Wahajul Haq
name: Himanshi Amita
name: Singh, Prince
name: Kiess, Wieland
name: Kaessner, Franziska
name: Garten, Antje
name: Smrati Bhadauria
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: Computational Biology

description: Additional file 1 : Figure S1. Superoxide anion generation in Pyrogallol treated cells. (A) MDA-MB-231 cells were treated with Pyrogallol (10, 20, 50 and 100 μM) for 24 h followed by 30 mins incubation with 10 μM DHE and analyzed for superoxide anion detection using a fluorescence microscope (above). Phase-contrast images of cells after indicated concentration of Pyrogallol treatment (below). Scale bars, 50 μm. (B) MDA-MB-231 cells were treated with 20 μM Pyrogallol for time point as indicated in the figure, and Western blotting was done for mTORC2 specific markers. All data are representative of three independent experiments. Figure S2. Pyrogallol prevents Ras localization to the plasma membrane. MDA-MB-231 cells pre-treated with FTI (Lonafarnib 1 μM) for 4 h followed by stimulation with Pyrogallol (20 μM) for another 24 h. Cells were analyzed for Ras localization by immunofluorescence microscopy. Data are representative of three independent experiments. Figure S3. Evaluation of sequences of peptides. Evaluation of sequences of synthetic peptides identical to the Ras-binding domain (RBD) of mSIN1. Figure S4. Peptide penetration and mutation analysis. (A) Immunofluorescence images of MDA-MB-231 cells treated with FITC-conjugated P4 (50 μg/ml) for 24 h. (B) RBD sequences of wild-type and mutant. Two of the amino acid residues Tyr-323(Y323) and Leu-325(L325) within the region corresponding to P4 in the wild-type RBD, were conserved across the species. The conserved amino acids tyrosine and leucine were mutated with alanine substitutions. Figure S5. Quantification of data of Fig. 1b Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 1b). **P ≤ 0.01, ***P ≤ 0.001. Figure S6. Quantification of data of Fig. 2c, d, and e. (A) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2c). (B) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2d). (C) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2e). Pyr (Pyrogallol) ns (not significant). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure S7. Quantification of data of Fig. 3a and f. (A) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data represented in Fig. 3a. (B) Densitometric quantification of Western blot data represented in Fig. 3f. VC (Vehicle control), Pyr (Pyrogallol), and ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure S8. Quantification of data of Fig. 4b and c. Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 4b). ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.