Additional file 1 of The mTOR pathway controls phosphorylation of BRAF at T401
Creators: name: Christen, Daniel
name: Lauinger, Manuel
name: Brunner, Melanie
name: Dengjel, Jörn
name: Brummer, Tilman
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1:  Figure S1. The monoclonal anti-pT401 antibody does not detect the BRAF T401A mutant. Western blot analysis of lysed Plat-E cells overexpressing HA-tagged BRAF WT or the T401A mutant, incubated with 1 µM torin1 or vehicle control for 4 h. Signals for pBRAF T401 and HA are shown. Figure S2. T401 phosphorylation inhibition by torin1 is concentration- and time-dependent, yet more stable than p70S6K phosphorylation. (A) Western blot analyses of a single titration experiment showing the concentration-dependent reduction of pT401 following torin1 treatment for 4 h. (B) Time course experiment demonstrating the dynamics of T401 phosphorylation loss upon torin1 treatment (1 µM). (C and D) Quantification of T401 phosphorylation intensities, normalized to total BRAF and a loading control (α-Tubulin) and related to the signal detected in the DMSO control. (E) T401 and p70S6K phosphorylation recover following torin1 washout. Western blot analysis of HEK293T cells inhibited with 1 µM torin1 for 4 h, then washed twice with ice-cold PBS, and incubated for the indicated time without an inhibitor prior to lysis. (F) Western blot analysis comparing the effects of different mTOR inhibitors, as well as afatinib and tacrolimus, on T401 phosphorylation. HEK293T cells were inhibited for 4 h before lysis and subjected to SDS-PAGE and Western blotting. Detection of tubulin (and vinculin) serves as loading control. Red colour indicates oversaturation of the imager. Figure S3. Quantification of BRAF phosphorylation sites using phosphoproteomics. Quantification of a SILAC-based mass spectrometry experiment. Braf−/− murine embryonic fibroblasts (MEFs) expressing ER T2 -HRAS G12V were transduced with human HA-tagged BRAF and treated for 24 h with either 4HT or ethanol (solvent control). Subsequently, cells were either incubated with torin1 or DMSO (vehicle) 4 h prior to lysis. Then, HA-tagged BRAF was immunoprecipitated, digested, and the abundance of the indicated phosphorylation sites was analysed. Shown is the fold change of one biological replicate of the phosphorylation of the indicated site of ER T2 -HRASG12V on vs. ER T2 -HRASG12V off (A) and of torin1/DMSO in the presence of ER T2-HRASG12V switched on (B). Error bars show the coefficient of variability over all redundant quantifiable peptides. (C) T401 peptide normalised occupancy was calculated for the three conditions as published previously [35]. Figure S4. BRAF T401 phosphorylation is neither enhanced by fetal calf serum nor EGF. (A) Western blot analysis of HEK293T cells that were incubated in medium containing the indicated percentage of FCS for 18 h. Samples in lanes 5 and 6 were derived from cells incubated in 1% FCS for 17 h followed by restimulation a higher FCS concentration (10% or 5%) for one hour. (B) HEK293T cells were pre-treated with either torin1 (500 nM), trametinib (Tram, 100 nM), or vehicle control for 4 h and then either left untreated or treated with hEGF (100 ng/mL) for 15 min. Detection of tubulin serves as a loading control. Figure S5. Constitutive Tsc1 deficiency does not affect BRAF T401 phosphorylation. Western blot analysis of TSC1 knock-out and control MEFs treated with either dactolisib (Da, 1 µM), trametinib (T, 100 nM) or DMSO (D) as a control for 4 h. Detection of tubulin serves as a loading control. Figure S6. BRAF co-purifies with Raptor and Rictor. (A) Co-immunoprecipitation of endogenous Raptor and Rictor with purified HA-tagged BRAF WT . (B) FACS Analysis of the generated Raptor and Rictor knock-down HEK293T cell lines. Cells were treated with 500 ng/mL doxycycline for 48 h before being subjected to FACS analysis. shRNA constructs additionally encode turboRFP to distinguish cells expressing the transduced shRNA constructs. Figure S7. BRAF T401A does not change mTORC1 signalling. Western Blot of HEK293T cells transfected with either pMIberry empty vector, HA-tagged BRAF wild-type or the BRAF T401A mutant. Lysates were prepared 48 h after transfection. Two independent biological replicates were used and membranes were probed with indicated antibodies. For the left replicate Tubulin, and for the right panel Vinculin served as loading control.