Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: De novo missense variants in RRAGC lead to a fatal mTORopathy of early childhood.

doi: 10.1016/j.gim.2023.100838

Figure Lengend Snippet: Figure 5 Subcellular localization of mTOR and biochemical analysis of published and novel variants in RRAGC. A. Patient (ID #1) and control (CT4) fibroblasts were stained with antibodies against mTOR and lysosomal-associated membrane protein 1 (Lamp1). Cells were cultured in the presence (+aa) or absence (−aa) of amino acids. Images show representative findings from 3 independent experiments. During the presence of amino acids, mTOR is mainly localized at the lysosomes. No clear differences between patient and control fibroblasts are visible under this condition. However, during amino acid starvation, mTOR redistributes to the cytosol in control fibroblasts. In contrast, mTOR remains mainly localized at the lysosomes in patient-derived fibroblasts. These findings suggest that the p.(Thr90Asn) variant de- couples mTOR activation from nutritional state. B. Representative western blot of protein lysates from HEK293 cells transfected with wild- type or mutant RRAGC incubated in media lacking L-leucine and L-methionine for 90 minutes. Whole-cell lysates were assessed for phosphorylated and total p70S6K and TFEB. B-actin was used as a loading control. The effect of the previously published variant p.(Ser75Tyr) was also assessed as a positive control. All novel variants were found to significantly increase S6 kinase and TFEB phos- phorylation compared with the wild type. C, D. Quantification of (C) phosphorylated relative to total p70S6K and (D) phosphorylated TFEB relative to total TFEB based on 3 independent biological replicates. Average values are presented as the mean ± SD. Significance was calculated using an ordinary one-way ANOVA in PRISM 9. *P < .05, **P < .01, ***P < .001, ****P < .0001. aa, amino acid; ANOVA, analysis of variance; mTOR, mechanistic target of rapamycin; TFEB, transcription factor EB.

Article Snippet: CoraLite 594- conjugated mTOR monoclonal antibody (mouse IgG2a; Proteintech CL594-66888; 1:100) and anti-LAMP1 monoclonal antibody (mouse IgG1, abcam ab25630; 1:100) were used as primary antibodies.

Techniques: Control, Staining, Membrane, Cell Culture, Derivative Assay, Variant Assay, Activation Assay, Western Blot, Transfection, Mutagenesis, Incubation, Positive Control

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison

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: Subsequently, the blocked membrane was incubated overnight at 4 °C with 1 μg of recombinant mTOR protein (Origene) per 1mL of 5% milk in TBST.

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: Subsequently, the blocked membrane was incubated overnight at 4 °C with 1 μg of recombinant mTOR protein (Origene) per 1mL of 5% milk in TBST.

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: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/bcj20230035

Figure Lengend Snippet: Figure 2. ABT-869 inhibits necroptosis in mouse and human cells. (A,B) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3

Article Snippet: Necroptosis-Inducing Stimuli Stimulus Component Supplier Concentration TSQ TNF Produced in-house [61] 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM Q-VD-OPh MP Biologicals 10 mM TSZ TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM z-VAD-fmk ApexBio 10 mM TSI TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM IDN-6556 (Emricasan) Tetralogic 5 mM Western Blot Primary Antibodies Protein Antibody Supplier Catalogue # Dilution Mouse MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Mouse RIPK1 Rabbit anti-RIPK1 Cell Signalling Technology cat #3493 1 : 1000 Mouse RIPK3 (CETSA) Rabbit anti-RIPK3 ProSci cat #2283 1 : 1000 Mouse RIPK3 (phosphorylation time course) Rat anti-mouse RIPK3 clone 8G7 Produced in-house [9], available from Merck Millipore cat #MABC1595 1 : 1000 Human MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Continued Figure 4.

Techniques: Control

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/bcj20230035

Figure Lengend Snippet: Figure 3. ABT-869 binds to RIPK1 in mouse and human cells. Part 1 of 2 (A) Binding affinities (KD) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by

Article Snippet: Necroptosis-Inducing Stimuli Stimulus Component Supplier Concentration TSQ TNF Produced in-house [61] 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM Q-VD-OPh MP Biologicals 10 mM TSZ TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM z-VAD-fmk ApexBio 10 mM TSI TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM IDN-6556 (Emricasan) Tetralogic 5 mM Western Blot Primary Antibodies Protein Antibody Supplier Catalogue # Dilution Mouse MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Mouse RIPK1 Rabbit anti-RIPK1 Cell Signalling Technology cat #3493 1 : 1000 Mouse RIPK3 (CETSA) Rabbit anti-RIPK3 ProSci cat #2283 1 : 1000 Mouse RIPK3 (phosphorylation time course) Rat anti-mouse RIPK3 clone 8G7 Produced in-house [9], available from Merck Millipore cat #MABC1595 1 : 1000 Human MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Continued Figure 4.

Techniques: Binding Assay

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/bcj20230035

Figure Lengend Snippet: Figure 4. ABT-869 binds to mouse and human RIPK1 in vitro. Part 1 of 2 Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869

Article Snippet: Necroptosis-Inducing Stimuli Stimulus Component Supplier Concentration TSQ TNF Produced in-house [61] 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM Q-VD-OPh MP Biologicals 10 mM TSZ TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM z-VAD-fmk ApexBio 10 mM TSI TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM IDN-6556 (Emricasan) Tetralogic 5 mM Western Blot Primary Antibodies Protein Antibody Supplier Catalogue # Dilution Mouse MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Mouse RIPK1 Rabbit anti-RIPK1 Cell Signalling Technology cat #3493 1 : 1000 Mouse RIPK3 (CETSA) Rabbit anti-RIPK3 ProSci cat #2283 1 : 1000 Mouse RIPK3 (phosphorylation time course) Rat anti-mouse RIPK3 clone 8G7 Produced in-house [9], available from Merck Millipore cat #MABC1595 1 : 1000 Human MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Continued Figure 4.

Techniques: In Vitro

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/bcj20230035

Figure Lengend Snippet: Figure 5. ABT-869 inhibits RIPK1 kinase activity in vitro and in cells. Part 1 of 2 (A–H) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase

Article Snippet: Necroptosis-Inducing Stimuli Stimulus Component Supplier Concentration TSQ TNF Produced in-house [61] 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM Q-VD-OPh MP Biologicals 10 mM TSZ TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM z-VAD-fmk ApexBio 10 mM TSI TNF Produced in-house 100 ng/ml Smac mimetic (Compound A) Tetralogic 500 nM IDN-6556 (Emricasan) Tetralogic 5 mM Western Blot Primary Antibodies Protein Antibody Supplier Catalogue # Dilution Mouse MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Mouse RIPK1 Rabbit anti-RIPK1 Cell Signalling Technology cat #3493 1 : 1000 Mouse RIPK3 (CETSA) Rabbit anti-RIPK3 ProSci cat #2283 1 : 1000 Mouse RIPK3 (phosphorylation time course) Rat anti-mouse RIPK3 clone 8G7 Produced in-house [9], available from Merck Millipore cat #MABC1595 1 : 1000 Human MLKL Rat anti-MLKL clone 3H1 Produced in-house [20], available from Merck Millipore cat #MABC604 1 : 1000 Continued Figure 4.

Techniques: Activity Assay, In Vitro, Phospho-proteomics