nras protein  (Proteintech)

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Analysis of response of cell lines from the Dependency Map portal (DepMap) database to CRISPR-knockout (blue curve) or RNAi-mediated inhibition of NRAS -mRNA (green curve) shows that the vast majority of cell lines presented no dependency on NRAS -mRNA expression (dependency score 0, black dotted line). b Filtering for melanoma cell lines showed that specifically NRAS -mutant melanoma cells presented a strong vulnerability on NRAS -mRNA expression (dependency score ≤ -1, red dotted line). Dot plots represent all analyzed cell lines (black: non-melanoma, yellow: NRAS wild type melanoma, red: NRAS -mutant melanoma), highlighting that the dependent melanoma cell lines harbor NRAS mutations. c Subcellular mRNA enrichment analysis was done using qRT-PCR to compare the ratio of nuclear versus cytoplasmic mRNA levels of NRAS , GAPDH and B-ACTIN in D04 and MM415 cells. The data are presented as fold-change of nuclear to cytoplasmic ratio normalized to GAPDH ( n = 3) and show higher nuclear enrichment of NRAS-mRNA , when compared to reference genes. The error bars represent Standard Error (s.e.m.). d , e Representative images of RNA in situ hybridization (RNA-ISH) derived from d D04 and e MM415 cell pellets. Fluorescent signals are either produced by DAPI DNA staining to mark the nuclear regions (blue) or probes that stain the NRAS -mRNA (red). f Quantification of punctua per nucleus from fluorescent signals produced by probes that stain NRAS -mRNA in D04 and MM415 cells. The calculations included > 1000 cells per cell line. g Intronic (small bars) and exonic (large bars) regions of the NRAS gene (ENSG00000213281.5) as annotated in the Genecode database (V44). NRAS ASO target regions are highlighted in black and the codons Q61 and G12 are highlighted in red. h NRAS -mRNA (Genecode ID: ENST00000369535.5) secondary structure as predicted by the Minimum Free Energy (MFE) model. NRAS ASO target regions are highlighted in black, provided in additional cutout and zoom. Codon Q61 is highlighted in red. The ASO target regions represent stable and accessible structures.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: CRISPR, Knock-Out, Inhibition, Expressing, Mutagenesis, Quantitative RT-PCR, RNA In Situ Hybridization, Derivative Assay, Produced, Staining

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Using qRT-PCR to compare RNA levels in D04 and MM415 cells that were either treated with NRAS ASO-1 or NRAS ASO-2, showed a robust reduction of NRAS -mRNA levels after 6, 24, 48, and 72 hours, when compared to treatment with non-targeting Control ASO. Final oligonucleotide concentration was 100 nM; error bars represent s.e.m. ( n = 3). b , c Representative images of RNA in situ hybridization (RNA-ISH) derived from pellets of b D04 or c MM415 cells, either treated with NRAS ASO-1, or Control ASO. Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange). NRAS ASO-1 treatment strongly reduced NRAS -mRNA levels in the cytoplasm and nucleus of the cells and NRAS protein expression. Final oligonucleotide concentration was 100 nM and treatment period lasted for 24 h. d Immunoblotting showing a strong decrease in NRAS protein levels 1 day after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−66%) and MM415 (−87%) cell lysates. B-ACTIN served as loading control and normalization parameter. e Immunoblotting showing a decrease in p-ERK1/2 protein levels 2 days after NRAS ASO treatment compared to Control ASO treatment in D04 (−50%) and MM415 (−50%) cell lysates, while total ERK1/2 levels were not altered significantly. GAPDH served as loading control and normalization parameter. f Immunoblotting showing a decrease in p-S6 protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−70%) and MM415 (−71%) cell lysates, while total S6 levels were not altered significantly. g Immunoblotting showing a small increase in p-AKT protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (+18%) and MM415 (+12%) cell lysates. Total AKT levels were not altered significantly. Final oligonucleotide concentration was 100 nM. h A simplified illustration depicting key signaling pathways in NRAS -mutant melanoma, emphasizing the activation of crucial proteins contributing to cellular survival. Through transcription, the mutations in the NRAS gene are carried over to the NRAS -mRNA, which is translated into the constitutively active mutant NRAS protein, initiating downstream signaling cascades. This activation prompts the RAF kinase (not shown) to activate MEK, which, in turn, activates ERK. ERK signaling influences the activation of S6 ribosomal protein and translocates to the nucleus, regulating transcription and supporting cellular proliferation. S6 plays a pivotal role in translation, facilitating protein synthesis. The activation of this signaling pathways enhances cellular survival in NRAS -mutant melanoma. Phosphorylation-dependent activation steps are denoted by (P). i A simplified illustration highlighting the impact of NRAS ASO treatment: NRAS ASOs reduce NRAS -mRNA levels in both the cytoplasm and nucleus. This reduction is followed by decreased NRAS protein levels and the inhibition of MAPK-pathway signaling activity, as evidenced by diminished p-ERK and p-S6 protein levels. The pathway is essential for the NRAS-mutant cancer cells’ ability to proliferate and survive.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Quantitative RT-PCR, Control, Concentration Assay, RNA In Situ Hybridization, Derivative Assay, Produced, Staining, Expressing, Western Blot, Protein-Protein interactions, Mutagenesis, Activation Assay, Phospho-proteomics, Inhibition, Activity Assay

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000002), MM415 ( p = 0.00002), WM1366 ( p = 0.0005), Sk-Mel-2 ( p = 0.00001), VMM39 ( p = 0.00004), WM3060 ( p = 0.003), NZM40 ( p = 0.0006), WM3629 ( p = 0.0008), and the primary derived cell line Hs852T ( p = 0.000006). b Treatment with NRAS ASO-2 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000004) and MM415 ( p = 0.0001). The antiproliferative outcomes are similar when compared to treatment with NRAS ASO-1. c NRAS ASO treatment did not cause significant antiproliferative effects in primary human melanocytes (PHM, p = 0.33), primary human liver cells (Hs775li, p = 0.29), human colon cells (FHC, p = 0.29), and BRAF-mutant melanoma cells (Sk-Mel-28, p = 0.13). d NRAS ASO treatment significantly inhibited colony formation in the D04 ( p = 0.0017) and MM415 ( p = 0.008) cell lines compared to treatment with non-targeting Control ASOs. Treatment period was 7 days (50 nM final oligonucleotide concentration, n = 3). e Representative images of D04 colonies in 6 cm dishes after ASO treatment. f Dot plot graph of flow cytometric analysis of PI and Annexin V staining after 1 day of ASO-treatment (100 nM) shows increased apoptotic cell death in D04-cells treated with NRAS ASO (15,780 total events) compared to Control ASO treatment (44,285 total events). g Distribution of overall cell populations from panel f ) in regards of their apoptotic state. Bars represent the percentage of vital (Q2), early apoptotic (Q3), late apoptotic (Q4) and dead (Q1) cells. h NRAS ASO-mediated induction of apoptosis was confirmed by measurement of significantly increased activity levels of the apoptosis markers Caspase-3 & -7 after 1 day of treatment with either NRAS or Control ASOs (100 nM) in the D04 ( p = 0.002) and MM415 ( p = 0.0002) cell lines ( n = 4). i Treatment with NRAS ASO−1 caused significant inhibition of cell growth in the NRAS -mutant multiple myeloma (MM) cell line H929 ( p = 0.0005), and small cell lung cancer (SCLC) cell line SW1271 ( p = 0.0001). j Significant tumor growth reduction was observed when comparing treatment groups for subcutaneous systemic treatment with either NRAS ASO (X) or Control ASO (O) in mouse models carrying xenografts of the D04 melanoma cell line (3 × 200 µg ASO/week, n = 6, days of measurement and p -values: −3 –0.38, 1 –0.27, 3 –0.02, 5 –0.04, 8 –0.05, 10 – 0.02, 12 –0.06, 15 –0.02, 17 – 0.02, 19 – 0.03). At the endpoint of the experiment (day 19), the average tumor size in the NRAS ASO treatment group was 48% smaller compared to control. k NRAS -mRNA levels were significantly reduced (0.68-fold, s.e.m = 0.03, p = 0.0003) in tumors of the NRAS ASO treatment group compared to the Control ASO treatment group at the end of study period. Tumors were harvested at end of treatment period; gene expression was normalized to Β-ACTIN expression and NRAS -mRNA expression in NRAS ASO treated tumors was normalized to expression in Control ASO treated tumors ( n of each group = 5). l No significant weight changes were observed between the NRAS ASO (X) and Control ASO (O) groups at any time-point (days of measurement and p -values: -3 – 0.3, 1 – 0.36, 3 – 0.33, 5 – 0.46, 8 – 0.43, 10 – 0.5, 12 – 0.47, 15 – 0.49, 17 – 0.48, 19 – 0.49). m Blood of mice that either received a dose of NRAS ASO-1 (200 µg/injection), or ASO-free PBS was drawn 24 hours after injection and analyzed for parameters of liver function (Serum transaminases – ALT, AST, bilirubin - TBIL, direct (conjugated) bilirubin - DBIL, total protein - TP, albumin - ALB, and alkaline phosphatase – ALKP). All growth and weight curves are presented as polynomial trend lines (order: 2). Data in ( a – c , i ) were normalized to treatment with non-targeting Control ASO, final oligonucleotide concentration was 50 nM, treatment period was 5 days ( n = 3). The error bars in a – d ), h , i , m ) represent s.d., in j − l they represent s.e.m. Significance is shown as p -values calculated by Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Inhibition, Mutagenesis, Derivative Assay, Control, Concentration Assay, Staining, Activity Assay, Gene Expression, Expressing, Injection

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Schematic illustration of HT-KAM analysis of the phosphor-catalytic activity of kinases. D04 and MM415 cells were either treated with NRAS or Control ASOs (50 nM, 1 day). Cells were lysed, and protein lysate was investigated for peptide-associated phosphorylation activity of kinases. b Comparison of kinase activity in treatment groups (NRAS ASO VS. Control ASO) showed that kinase activity of several kinases was significantly upregulated in the D04 and MM415 cell lines upon NRAS ASO treatment. Kinases are ranked by their relative increase of activity (from bottom to top). The top 3 kinases with strongest shift in activity increase are MAP2K1 (MEK1), FGFR2, and CDK4. The RET kinase activity shift is shown as a representative example for kinases that were downregulated in activity. c QRT-PCR analysis showing elevated NRAS -mRNA levels in D04 and MM415 cells after three days of drug-induced Inhibition of MEK (MEKi), using the small molecule inhibitor Trametinib (20 nM or 40 nM), when compared to control, treated with DMSO ( n = 3). d QRT-PCR analysis showing elevated NRAS -mRNA levels in the MEKi resistant cell lines D04RM and MM415RM, which were chronically exposed to Trametinib, when compared to their paternal treatment naïve cell lines D04 and MM415 ( n = 3). Error bars in panel ( c , d ) represent s.e.m. e Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the MEKi resistant NRAS mutant melanoma cell lines D04RM (p = 0.011), MM415RM ( p = 0.001), WM3629RM ( p = 0.0002), and Sk-Mel-2RM ( p = 0.015). Data were normalized to treatment with non-targeting Control ASO; treatment period was 5 days, final oligonucleotide concentration was 50 nM, and error bars represent s.d. ( n = 3). f – i Dual treatment with 20 nM of NRAS ASO and Trametinib (Tram, 0.5 nM −25 nM) caused robust synergistic effects in D04 ( f , g ) and MM415 ( h , i ) cells after 3 ( f , h ) and 5 ( g , i ) days of treatment ( n = 2). Dose response curves show NRAS ASO treatment (blue), trametinib treatment (yellow) and dual treatment (red). Synergism of dual cell growth inhibition is shown as bar graphs and determined by the HSA synergy score.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Activity Assay, Control, Phospho-proteomics, Comparison, Quantitative RT-PCR, Inhibition, Mutagenesis, 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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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) Proximity-dependent protein labeling (BioID) experimental workflow. LC-MS/MS, Liquid chromatography tandem-mass spectrometry. PSM, peptide-spectrum match. (B) Overlap of Ras-proximal proteins identified in each birA*:Ras isoform interactome. SAINT probability score ≥ 0.8; fold change ≥ 4 over birA* control. (C) Interaction network of 150 proteins common to all Ras isoforms. P-value calculated via STRING (Szklarczyk et al., 2015). (D) New and known interactors within 150 common proteins. (E) PLA in MT NRAS MM415 melanoma cells of endogenous Ras interactome candidates with endogenous Ras protein; punctate signal as reported (red) (Patricelli et al., 2016), nuclei (blue). None is single antibody control. Scale bar, 20μm. (F) Quantification of the PLA analysis shown in (E). n= 4-5 fields per sample, 1 experiment; mean ± SEM. **p< 0.01, ****p< 0.0001 (unpaired two-sided t-test to single antibody controls). See also Figures S1 and S2, and Table S1.

Article Snippet: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Control

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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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) GSEA enrichment plots of genes altered with MAPKAP1Del expression in MT versus WT NRAS melanoma cell lines. NES, normalized enrichment score. (B) GSEA enrichment plot of MT NRAS cell line-derived MAPKAP1Del gene signature in MT versus WT NRAS TCGA melanoma patient samples. (C) Day 0 normalized bioluminescence for empty vector (EV), MAPKAP1WT:FHH or MAPKAP1Del:FHH expressing MM485 MT NRAS melanoma tumors at final time point. n=4 mice/group, one experiment; (unpaired two-sided t-test). (D) Representative images of tumors from experiment in (C). (E) Western blot of EV, MAPKAP1WT:FHH, or MAPKAP1Del:FHH-expressing MM485 tumors. (F) Quantification of western blots shown in (E) normalized to total protein. (unpaired two-sided t-test). All data are mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, and ns= not significant. See also Figure S7, Table S6.

Article Snippet: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Western Blot

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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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: (A) Western blot of replicate in vitro mTORC2 IP-kinase assays with a GST-Akt1 tail substrate. Input levels right panel. HA-tagged MAPKAP1 pulldown of mTORC2 from 293T cells co-expressing empty vector (EV) or FLAG-GTPase. 5 μM PP242 (mTORi). (B) Quantification of the GST normalized mTORC2 IP-kinase assay pAktS473 signal, as in (A), relative to mean of the untreated EV control. n= 4-6 (unpaired two-sided t-test). (C) Quantification of HA IP mTORC2 kinase assay with addition of indicated recombinant GDP-loaded WT or GTPγS-loaded G12V H-Ras in vitro relative to EV in buffer only. n=5-6; ** p < 0.01, (unpaired two-sided t-test). (D) Diagram of the LocaTOR2 assay for subcellular compartment-specific in vivo mTORC2 kinase activity. (E) LocaTOR2-based subcellular compartment mTORC2 activity quantified as phosphorylated Frb:Akt2S474 signal over untreated in MT NRAS MM485 melanoma cells. 500 nM KU-0063794 (Pan-mTORi). Dotted line at 2. n=2 experimental replicates. (F) Quantification of compartment-specific LocaTOR2 signal with shSCR or shNRAS knockdown, n=3. (Welch’s two-sided t-test to respective shSCR). (G) Quantification of protein remaining after knockdown compared to shSCR for (F). All data are mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001. See also Figure S6.

Article Snippet: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

Techniques: Western Blot, In Vitro, Expressing, Plasmid Preparation, IP-Kinase Assay, Control, Kinase Assay, Recombinant, In Vivo, Activity Assay, Knockdown

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: NRAS Q61K Human Recombinant Protein , Origene , Cat# TP701004.

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