Journal: Archives of Medical Science : AMS

Article Title: Efficacy of Setarud (IMOD™), a novel electromagnetically-treated multi-herbal compound, in mouse immunogenic type-1 diabetes

doi: 10.5114/aoms.2010.17078

Figure Lengend Snippet: Pancreatic level of oxidative stress markers in experimental groups on day 21. IMOD™ (20 mg/kg/d, IP) decreased MPO activity ( A ) and lipid peroxidation, assessed by TBARS concentration ( B ), in pancreas samples of treated mice. Total antioxidant power (FRAP) of pancreatic tissue increased in IMODTM treated groups ( C ) *p < 0.01 comparing with ND-C; **p < 0.01 comparing with D-C

Article Snippet: Materials for measurement of thiobarbituric acid reactive substances (TBARS), total antioxidant power (ferric reducing ability of pancreas; FRAP), myeloperoxidase (MPO), protein, and sample preparation for histological assay were purchased from Merck Chemical Co. (Tehran).

Techniques: Activity Assay, 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: (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: For Ras effector Far Western experiments, the indicated amount of MAPKAP1 (Origene), mTOR (Origene), PI3Kγ (Origene), RalGDS (Origene), RAF1 (Origene), GST (Millipore Sigma), mLST8 (Abnova) or Akt (Jena Bioscience) were spotted onto nitrocellulose membrane and allowed to air dry.

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: For Ras effector Far Western experiments, the indicated amount of MAPKAP1 (Origene), mTOR (Origene), PI3Kγ (Origene), RalGDS (Origene), RAF1 (Origene), GST (Millipore Sigma), mLST8 (Abnova) or Akt (Jena Bioscience) were spotted onto nitrocellulose membrane and allowed to air dry.

Techniques: Binding Assay, Microscale Thermophoresis, Labeling, Titration, Western Blot, 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: For Ras effector Far Western experiments, the indicated amount of MAPKAP1 (Origene), mTOR (Origene), PI3Kγ (Origene), RalGDS (Origene), RAF1 (Origene), GST (Millipore Sigma), mLST8 (Abnova) or Akt (Jena Bioscience) were spotted onto nitrocellulose membrane and allowed to air dry.

Techniques: RNA Expression, Western Blot, RNA Sequencing Assay, Labeling, Derivative Assay, 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: For Ras effector Far Western experiments, the indicated amount of MAPKAP1 (Origene), mTOR (Origene), PI3Kγ (Origene), RalGDS (Origene), RAF1 (Origene), GST (Millipore Sigma), mLST8 (Abnova) or Akt (Jena Bioscience) were spotted onto nitrocellulose membrane and allowed to air dry.

Techniques: Western Blot, 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: For Ras effector Far Western experiments, the indicated amount of MAPKAP1 (Origene), mTOR (Origene), PI3Kγ (Origene), RalGDS (Origene), RAF1 (Origene), GST (Millipore Sigma), mLST8 (Abnova) or Akt (Jena Bioscience) were spotted onto nitrocellulose membrane and allowed to air dry.

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