mtor human sirna oligo duplex  (OriGene)

Journal: Nature Communications

Article Title: mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1

doi: 10.1038/s41467-022-32487-7

Figure Lengend Snippet: a Schematic representation of SCYL1 protein structure. Known conserved domains and phosphorylation sites identified by MS are shown. CC = Coiled-coil domain. Black stars: phosphorylation events detected in independent AP-MS experiments; Red star: Ser754, identified reproducibly as regulated in in vitro mTOR kinase assays. Blue lollipop chart: number of citations referenced in Phosphosite Plus® for indicated phosphosites. Pink lollipop chart: functional score for indicated phosphosites, as published in ref. . b SCYL1 Ser754 is rapidly dephosphorylated upon mTORC1 inhibition. Phosphoproteomic time-course of SCYL1 Ser754 phosphorylation levels in SILAC-labeled MCF-7 WT cells upon HBSS starvation or 100 nM rapamycin treatment. Average values of n = 2 biological replicates. c In vitro mTOR kinase assay qualifies SCYL1 Ser754 as direct mTOR target site. Purified mTOR was incubated with immunoprecipitated HA-SCYL1 in absence or presence of wortmannin (12 µM, 30 min). Two biological replicates were analyzed by LC-MS/MS. Results are displayed as a ratio of phosphosite intensities without/with wortmannin (Supplementary Data ). d Time-course of SCYL1 Ser754 phosphorylation upon mTORC1 inhibition, detected by a phosphosite-specific antibody. WT MCF-7 cells were left untreated or treated with 100 nM rapamycin, starvation (HBSS), or 250 µM Torin1, for the indicated times. One representative time-course experiment out of three biological replicates is shown. Equal protein amounts were loaded. e Quantification of d . Phospho-Ser754-SCYL1 levels were normalized to total SCYL1 levels. Average of n = 4 biological replicates for DMEM/Rapa and DMEM/Starv measurements, and n = 3 biological replicates for DMEM/Torin1 measurements. Red line: rapamycin/DMEM 0 min; blue line: starvation/DMEM 0 min; green lines: Torin1/DMEM 0 min. All single values are indicated by colored dots. p -values: An unpaired two-tailed Student’s t -test was used to compare the indicated time points with the 0 min reference; Rapa10’/DMEM p = 0.0068(**); Rapa 30’/DMEM p = 0.017(*); Rapa 60’/DMEM p = 0.018(*); HBSS 60’/DMEM p = 0.038(*); Torin10’/DMEM p = 0.0029(**); Torin30’/DMEM p = 0.049(*); Torin60’/DMEM p = 0.043(*). Error bars, SEM. f SCYL1 Ser754 phosphorylation responds to mTORC1 reactivation. WT MCF-7 cells were starved in HBSS for 4 h, then released in complete DMEM supplemented with 50 µg/ml cycloheximide to increase the intracellular amino acid concentration for the indicated times. One representative time-course experiment out of three biological replicates is shown. Equal protein amounts were loaded. g Quantification of f . Phospho-Ser754-SCYL1 levels were normalized to total SCYL1 levels. Average of n = 3 biological replicates. p -values: An unpaired two-tailed Student’s t -test was used to compare the indicated time points with the starved cells reference time point (before release); 30’: p = 0.0028(**); 60’: p = 0.0018(**). Error bars, SEM. All source data are provided as Source Data file.

Article Snippet: The commercial mTOR (TP320457, ORIGENE), or the in house -purified MTORC1 complex on anti-FLAG M2 affinity gel, and the HA-SCYL1-coated beads were added onto a 10 kD MW-cutoff filter (Sartorius) in 100 μl of kinase buffer with 1 mM γ-[ 18 O 4 ]-ATP (Cambridge Isotope Laboratory, OLM-7858), and for the the in house -purified MTORC1 assay 100 μg/ml of Flag peptide (Sigma, F3290) to elute the complex.

Techniques: Phospho-proteomics, Protein-Protein interactions, In Vitro, Functional Assay, Inhibition, Multiplex sample analysis, Labeling, Kinase Assay, Purification, Incubation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Concentration Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Western Blot, Transduction, In Situ, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Clone Assay, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing Assay, Sequencing, Expressing, CRISPR, Mass Spectrometry, Mutagenesis, shRNA, Plasmid Preparation, Luciferase, Software, Blocking Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) A two-week growth CRISPR screen of 150 common Ras-proximal proteins in diploid primary human melanocytes (MC) and 5 MT Ras cell lines. Left heatmap negative selection FDR values (FDR ≤ 0.18) in each cell type by the MAGeCK algorithm (Li et al., 2014). The right heatmap relative enrichment MT vs WT Ras in mass spectrometry data for each Ras isoform; new proteins (red), known proteins (*). Ranked by combined FDR and log2(PSM MT/WT Ras) score. (B) Common Ras-proximal interacting protein-protein network based on candidates with ≥1 database interaction with another common interactor. Large squares are novel and small squares are known interactors. (C) PLA in MT NRAS MM415 melanoma cells with endogenous mTOR and Pan-Ras or Ras:GTP; interaction (red), nuclei (blue). Scale bar, 20 μm. (D) Quantification of PLA analysis in (C). n=8-10 fields/condition. (E) Western blot of HA co-immunoprecipitation of empty vector (EV), FLAG-HA-6xHIS tagged NRASWT or FHH: NRASQ61K with endogenous mTOR, p110α and Raf1 in wild-type RAS CHL-1 cells. (F) Quantification of HA co-IP experiments as in (E). Values are normalized to HA pulldown signal and relative to NRASWT signal, n= 6 (Wilcoxon Signed Rank Test). (G) PLA in genotyped human colorectal adenocarcinomas with endogenous Pan-Ras and mTOR. Scale bar, 20 μm. (H) Quantification of PLA analysis in (G). Each dot represents median signal per patient. n=5 patients/genotype group, ≥7 images analyzed per patient (Mann-Whitney U Test). (I) Microscale thermophoresis with labeled FHH:Raf1RBD (18.2nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is negative as the MST signal of the complex is lower than that of Raf1RBD alone. MST-on time of 15s, n = 3 independent replicates. (J) Microscale thermophoresis with labeled FHH:mTORKinaseDomain and FHH:mTORHEAT (23.4nM) with a titration series of GDP or GTPγs-loaded Ras. MST-on time of 2.5s, n ≥ 3 independent replicates. MST is mean ± SD. All other data mean ± SEM; *p< 0.05. See also Figures S3 and S4, Tables S2–4.

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: CRISPR, Selection, Mass Spectrometry, Western Blot, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay, MANN-WHITNEY, Microscale Thermophoresis, Labeling, Titration, Binding Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) Schematic of full length MAPKAP1 isoform 1 wild-type (WT) and the MAPKAP1 deletion (Del) proteins with domains highlighted. CRIM, Conserved Region In The Middle. RBD, Ras Binding Domain. PH, Pleckstrin Homology. (B) Microscale thermophoresis of labeled FHH:MAPKAP1RBD (16.8nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is positive as the MST signal of the complex is higher than MAPKAP1RBD alone. MST-on time of 5s, n = 3 independent replicates. (C) BioID-western blot streptavidin pulldowns and input levels for birA* control, NRASWT, NRASQ61K and NRASQ61K with FHH:eGFP, MAPKAP1WT:FHH or MAPKAP1Del:FHH expression in CHL-1 cells. PI3K p110α subunit, Raf-1, and HA protein pulldown are controls. Pulldown normalized signal relative to control birA*:NRASQ61K shown below. (D) Quantification of mTOR and Rictor protein levels in the streptavidin pulldowns normalized to HA pulldown. All values and statistical tests relative to birA*:NRASQ61K, n=6 (Welch’s two-sided t-test). (E) PLA with endogenous Pan-Ras and mTOR or Rictor in MT NRAS MM485 melanoma cells. Scale bar, 20 μm. (F) PLA quantification in (E). n=3 independent experiments, 6-8 fields analyzed per condition per experiment (unpaired two-sided t-test). EV, empty vector. (G) Quantification of LocaTOR2 experiments with FHH:eGFP, MAPKAP1WT:FHH or MAPKAP1Del:FHH expression. All values relative to average of FHH:eGFP and MAPKAP1WT:FHH fold induction; n=3 (unpaired two-sided t-test). (H) Quantification of FHH:eGFP, MAPKAP1WT:FHH and MAPKAP1Del:FHH expression relative to MAPKAP1WT:FHH for all experiments graphed in (G). **p< 0.01, ***p< 0.001, ****p< 0.0001, and ns= not significant; all bar graphed data mean ± SEM. MST data are mean ± SD. See also Figure S6 and Table S4.

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Binding Assay, Microscale Thermophoresis, Labeling, Titration, Western Blot, Control, Expressing, Plasmid Preparation

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of RICTOR (Normalized enrichment score = −1.20) and RPTOR (NES= −1.30) knockdown signatures from primary human melanocytes comparing NRASWT versus NRASMT melanoma patient sample TCGA RNA expression data. *p< 0.05. (B) Western blot of protein remaining in control, RPTOR, RICTOR, MAPKAP1, and NRAS knockdown RNA-sequencing samples in MT NRAS melanoma cell line MM485. Labeled with normalized protein remaining relative to the control average. (C) Heat map showing log2(fold change) in RNA-sequencing of MT NRAS melanoma line, MM485, with RICTOR, MAPKAP1, RPTOR or NRAS knockdown compared to control. mTORC2 gene signature is derived from concordant genes between shRICTOR and shMAPKAP1 samples and mTORC1 signature between shRPTOR samples. (D) Heatmap of −log10(adjusted p-values) for top GO terms for NRAS-regulated shMTORC1 and shMTORC2 down regulated gene sets as labeled in (C). Unfiltered shNRAS down regulated gene set analysis for comparison. (E) Heatmap of −log10(FDR q-values) for GSEA positively enriched REACTOME or KEGG or (F) Pathway interaction database (PID) signatures from Molecular Signatures Database with MTOR, RICTOR, MAPKAP1 or RPTOR expression as the phenotype in MT NRAS TCGA melanoma patient samples. Relevant signatures (red). See also Figure S7, Tables S5 and S6.

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Knockdown, RNA Expression, Western Blot, Control, RNA Sequencing, Labeling, Derivative Assay, Comparison, Expressing

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) BioID-western blot showing mTOR protein levels in the streptavidin pulldowns of birA*:NRASQ61K with shGFP, shRPTOR, shRICTOR or shMAPKAP1 with two independent hairpins in CHL-1 cells. PI3K p110α subunit, Raf1, and HA protein levels in streptavidin pulldowns are controls. Pulldown and input normalized values relative to the control shown below. (B) Quantification of mTOR, p110α and Raf1 protein levels in the streptavidin pulldown Normalized to HA pulldown and respective input levels and relative to shGFP mean, n=5 (unpaired two-sided t-test). (C) Quantification of protein remaining after knockdown compared to the average of controls for (B). (D) PLA in MT NRAS MM415 melanoma cells with endogenous Ras and mTOR with control, mTORC1 or mTORC2 component knockdown. Scale bar, 20 μm. (E) Quantification of PLA shown in (D). n=2 independent hairpins per knockdown. Relative to the mean of control knockdowns. (unpaired two-sided t-test). (F) Quantification of protein remaining after knockdown relative to mean signal of control knockdowns for PLA in (E). (G) Western blot of HA co-immunoprecipitation of empty vector (EV), FHH:NRASWT or FHH:NRASQ61K with endogenous Rictor, MAPKAP1 and Raptor in wild-type Ras CHL-1 cells. (H) Quantification of HA co-IP experiments as in (G). Values normalized to HA pulldown signal and relative to NRASWT signal. n= 5 or 8 (Welch’s two-sided t-test). (I) Western blot showing mTOR protein levels in the input and streptavidin pulldowns of birA* control, NRASWT, NRASQ61K and NRASQ61K Effector Domain Alanine point mutants. (*) non-specific background band. (J) Quantification of streptavidin pulldown protein levels as in (I). mTOR signal normalized to HA signal. All values relative to birA*: NRASQ61K, n=3 (Welch’s two-sided t-test relative to Q61K). All graphed data are mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, ns= not significant. See also Figure S5.

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Western Blot, Control, Knockdown, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Western Blot, Transduction, In Situ, Virus, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Cloning, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing, Sequencing, Gene Expression, CRISPR, Mass Spectrometry, Expressing, Mutagenesis, shRNA, Plasmid Preparation, Control, Luciferase, Software, Membrane, Blocking Assay