mtor total  (Cell Signaling Technology Inc)

Journal: The Journal of Nutrition

Article Title: Amino Acid Signaling in Skeletal Muscle Is Blunted by Prematurity in a Piglet Model

doi: 10.1016/j.tjnut.2025.101303

Figure Lengend Snippet: Current concepts of multiple amino acid–sensing input to mTORC1. AA, amino acid; Akt, RAC-α serine/threonine-protein kinase; ARF1, ADP-ribosylation factor 1; BCAA, branched-chain amino acid; CASTOR1, cytosolic arginine sensor for mTORC1 subunit 1; GATOR1/2, GAP activity toward RAGs 1/2; LARS, leucine-tRNA-synthase; LAT1/SLC7A5, L-type amino acid transporter 1; mTORC1, mechanistic target of rapamycin complex 1; PI3K, phosphatidylinositol-3-kinase; RAB1A, Ras-related protein Rab-1A; Rag A/B C/D, RAS-related GTP-binding protein A/B C/D; Ragulator, Late endosomal/lysosomal adaptor and MAPK and mTOR activator; Rheb, Ras homolog enriched in brain; SAM, S-adenosyl methionine; SAMTOR, S-adenosylmethionine sensor upstream of mTORC1; SAR1B, secretion-associated Ras-related GTPase 1B; Sestrin1/2, stress response protein 1/2; SLC38A9, solute carrier family 38 member 9; SNAT2, sodium-coupled neutral amino acid transporter 2; TARS2, threonyl-tRNA synthetase 2; TFEB, transcription factor EB; TSC1/2, tuberous sclerosis complex 1/2; V-ATPase, vacuolar H + -ATPase. Created in BioRender. Ramos dos Santos, A.C. (2026) https://BioRender.com/54b947z .

Article Snippet: Immunoblotting and immunoprecipitation assays were performed using the following primary antibodies: ARF1 (Cat. No. 68069-1-AP; ProteinTech Group), CASTOR1/GATSL3 (Cat. No. A13309; Boster Biological Technology), LARS (Cat. No. A304-316A; Bethyl Laboratories), Mios, a GATOR2 subunit (Cat. No. 13557; Cell Signaling Technology), mTOR phosphorylated Ser2448 (Cat. No. 2971; Cell Signaling Technology), mTOR total (Cat. No. 2972; Cell Signaling Technology), NPRL2, a GATOR1 subunit (Cat. No. 37344, 37344; Cell Signaling Technology), Rab1A (Cat. No. 13075; Cell Signaling Technology), RagA (Cat. No. 4357; Cell Signaling Technology), RagC (Cat. No. 9480; Cell Signaling Technology), Raptor (Cat. No. 2280; Cell Signaling Technology), SAMTOR/BMT2 (Cat. No. 21744-1-AP; ProteinTech Group), SAR1B (Cat. No. 22292-1-AP; ProteinTech Group), Sestrin1 (Cat. No. 55010-1-AP; ProteinTech Group), Sestrin2 (Cat. No. 66297-1-Ig; ProteinTech Group), SLC38A2/sodium-coupled neutral amino acid transporter (SNAT)2 (Cat. No. ARP33059_P050; Aviva Systems Biology), SLC38A9 (Cat. No. AAS59532C; Antibody Verify), LAT/1SLC7A5 (Cat. No. BMP011; MBL International), and TARS2 (Cat. No. 15067-1-AP; ProteinTech Group).

Techniques: Activity Assay, Binding Assay

Journal: iScience

Article Title: The ionotropic AMPA receptor contributes to autoimmunity via altered regulatory T cell differentiation

doi: 10.1016/j.isci.2025.114267

Figure Lengend Snippet: AMPAR-deficient CD4 T cells changed cytokine sensitivity and metabolically shifted toward glycolysis and lipid metabolism (A) Quantification of Foxp3 gMFI flow staining of WT and T ΔAMPAR iTregs cultured under the titration of IL-2 (mean ± SEM, ANOVA, representative of 3 independent experiments). (B) Representative flow plot of pSTAT5 + Foxp3 + expression (left) and quantification (right) from WT and T ΔAMPAR iTreg cultures (mean ± SEM, t test, representative of 3 independent experiments). (C) Quantification of CD4 + CD25 + frequency over the course of iTreg differentiation (mean ± SEM, ANOVA, representative of 3 independent experiments). (D) Western blot of WT and T ΔAMPAR CD4 T cells collected on day 1, 2, and 3 of iTreg differentiation, immunoblotted for phosphorylated mTOR (pmTOR), total mTOR, and β-Actin expression (top to bottom, representative of 3 independent experiments). (E) Quantification of CD71 + cells among CD4 + T cells on day 3 of iTreg differentiation. (mean ± SEM, t test, representative of 3 independent experiments). (F–I) Seahorse metabolic flux analysis of iTreg cells. (F) extracellular acidification rate (ECAR) measurement over time (minutes) and (G) quantification of glycolysis (left) and glycolytic capacity (right) measurements. (H) oxygen consumption rate (OCR) measurement over time (minutes) and (I) quantification of maximum respiratory capacity (left) and reserve capacity (right) measurements (mean ± SEM, ANOVA, representative of 3 independent experiments). (J and K) WT and T ΔAMPAR CD4 were cultured under iTreg polarizing conditions and subjected to bulk RNA sequencing and analysis. (J) GSEA plot of cholesterol metabolism pathway enrichment in T ΔAMPAR versus WT cells. (K) Heatmap of genes associated with cholesterol metabolism in T ΔAMPAR versus WT. (L) Quantification of Srebf1 , Srebf2 , Hmgcr , Acaca , Fasn , and Sqle mRNA relative expression in WT and T ΔAMPAR CD4 from iTreg culture determined by qPCR (mean ± SEM, ANOVA, representative of 2 independent experiments). (M) Western blot images (left) of WT and T ΔAMPAR immunoblotted for Srebf1 and Srebf2 (image is representative of 2 independent experiments) and quantification (right) of cleaved to precursor ratios analyzed by densitometry. (N and O) Cell staining for Lipid and cholesterol content. (N) Bodipy and (O) Filipin III gMFI flow staining of WT and T ΔAMPAR CD4 from day 2 iTreg cultures (mean ± SEM, t test, representative of 3 independent experiments). Statistical significance represented as ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Probes included: anti-pSMAD3 (Cell signaling, 9520), anti-total SMAD2/3 (Cell signaling, 3102), anti-pmTOR (Cell signaling, 2971), anti-total mTOR (Cell signaling, 2983), anti-Srebp1 (Abcam, ab3259), anti-Srebp2 (Abcam, ab30682), anti-β-tubulin (Cell signaling, 86298) and anti-β-actin (Cell signaling, 4970).

Techniques: Metabolic Labelling, Staining, Cell Culture, Titration, Expressing, Western Blot, RNA Sequencing

Journal: Cancers

Article Title: Chondrosarcoma: Multi-Targeting Therapeutic Effects of Doxorubicin, BEZ235, and the Small Molecule Aspartyl-Asparaginyl-β-hydroxylase Inhibitor SMI1182

doi: 10.3390/cancers17101671

Figure Lengend Snippet: Effects of SMI1182 (SMI), DOX, and BEZ on the expression of mid-level components of the iInsulin/IGF1-Akt-mTOR pathway in CS1 cells. Magnetic bead-based protein and phosphoprotein 11-plex ELISAs measured frontal lobe levels of ( A ) Akt, ( B ) GSK-3α, ( C ) GSK-3β, ( D ) PTEN, ( E ) pS473-Akt, ( F ) pS21-GSK-3α, ( G ) pS9-GSK-3β, ( H ) pS380-PTEN, and the calculated levels of relative phosphorylation of ( I ) p/T-Akt, ( J ) p/T-GSK-3α, and ( K ) p/T-GSK-3β, and ( L ), and p/T-PTEN, (compared with total protein) in cells (n = 4/group) treated for 48 h with Veh, SMI, DOX, or BEZ. Graphed values correspond to arbitrary fluorescent light units (FLU). Inter-group comparisons were made using two-way ANOVA tests with post -hoc Tukey tests. Software-generated p -values corresponding to significant differences ( p ≤ 0.05) are shown in the panels.

Article Snippet: We used 11-Plex MILLIPLEX Akt/mTOR Total and Phosphoprotein Magnetic Bead Kits (MilliporeSigma, Burlington, MA, USA) to evaluate the effects of treatment on mTOR signaling mechanisms in CS cells ( ).

Techniques: Expressing, Phospho-proteomics, Software, Generated

Journal: Cancers

Article Title: Chondrosarcoma: Multi-Targeting Therapeutic Effects of Doxorubicin, BEZ235, and the Small Molecule Aspartyl-Asparaginyl-β-hydroxylase Inhibitor SMI1182

doi: 10.3390/cancers17101671

Figure Lengend Snippet: Effects of SMI1182 (SMI), DOX, and BEZ on the expression of mid-level components of the iInsulin/IGF1-Akt-mTOR pathway in CDS11 cells. Magnetic bead-based protein and phosphoprotein 11-plex ELISAs measured frontal lobe levels of ( A ) Akt, ( B ) GSK-3α, ( C ) GSK-3β, ( D ) PTEN, ( E ) pS473-Akt, ( F ) pS21-GSK-3α, ( G ) pS9-GSK-3β, ( H ) pS380-PTEN, and the calculated levels of relative phosphorylation of ( I ) p/T-Akt, ( J ) p/T-GSK-3α, and ( K ) p/T-GSK-3β, and ( L ), and p/T-PTEN, (compared with total protein) in cells (n = 4/group) treated for 48 h with Veh, SMI, DOX, or BEZ. Graphed values correspond to arbitrary fluorescent light units (FLU). Inter-group comparisons were made using two-way ANOVA tests with post -hoc Tukey tests. Software-generated p -values corresponding to significant differences ( p ≤ 0.05) are shown in the panels.

Article Snippet: We used 11-Plex MILLIPLEX Akt/mTOR Total and Phosphoprotein Magnetic Bead Kits (MilliporeSigma, Burlington, MA, USA) to evaluate the effects of treatment on mTOR signaling mechanisms in CS cells ( ).

Techniques: Expressing, Phospho-proteomics, Software, Generated

Journal: Cancers

Article Title: Chondrosarcoma: Multi-Targeting Therapeutic Effects of Doxorubicin, BEZ235, and the Small Molecule Aspartyl-Asparaginyl-β-hydroxylase Inhibitor SMI1182

doi: 10.3390/cancers17101671

Figure Lengend Snippet: Effects of SMI1182 (SMI), DOX, and BEZ on the expression of downstream signaling through mTOR in CS1 cells. Magnetic bead-based protein and phosphoprotein 11-plex ELISAs measured immunoreactivity to ( A ) TSC2, ( B ) mTOR, ( C ) RPS6, ( D ) P70S6K, ( E ) pS939-TSC2, ( F ) pS2448-mTOR, ( G ) pS235/236-RPS6, ( H ) pT412-P70S6K, and the calculated relative levels of signaling molecule phosphorylation of ( I ) TSC, ( J ) mTOR, ( K ) RPS6, and ( L ) P70S6K in cells (n = 4 cultures/group) treated for 48 h with Veh, SMI, DOX, or BEZ. Graphed values correspond to arbitrary fluorescent light units (FLU). Inter-group comparisons were made by a two-way ANOVA . Software-calculated p -values reflecting significant differences ( p ≤ 0.05) are shown in the panels.

Article Snippet: We used 11-Plex MILLIPLEX Akt/mTOR Total and Phosphoprotein Magnetic Bead Kits (MilliporeSigma, Burlington, MA, USA) to evaluate the effects of treatment on mTOR signaling mechanisms in CS cells ( ).

Techniques: Expressing, Phospho-proteomics, Software

Journal: Cancers

Article Title: Chondrosarcoma: Multi-Targeting Therapeutic Effects of Doxorubicin, BEZ235, and the Small Molecule Aspartyl-Asparaginyl-β-hydroxylase Inhibitor SMI1182

doi: 10.3390/cancers17101671

Figure Lengend Snippet: Effects of SMI1182 (SMI), DOX, and BEZ on the expression of downstream signaling through mTOR in CDS11 cells. Magnetic bead-based protein and phosphoprotein 11-plex ELISAs measured immunoreactivity to ( A ) TSC2, ( B ) mTOR, ( C ) RPS6, ( D ) P70S6K, ( E ) pS939-TSC2, ( F ) pS2448-mTOR, ( G ) pS235/236-RPS6, ( H ) pT412-P70S6K, and the calculated relative levels of signaling molecule phosphorylation of ( I ) TSC, ( J ) mTOR, ( K ) RPS6, and ( L ) P70S6K in cells (n = 4 cultures/group) treated for 48 h with Veh, SMI, DOX, or BEZ. Graphed values correspond to arbitrary fluorescent light units (FLU). Inter-group comparisons were made by a two-way ANOVA . Software-calculated p -values reflecting significant differences ( p ≤ 0.05) are shown in the panels.

Article Snippet: We used 11-Plex MILLIPLEX Akt/mTOR Total and Phosphoprotein Magnetic Bead Kits (MilliporeSigma, Burlington, MA, USA) to evaluate the effects of treatment on mTOR signaling mechanisms in CS cells ( ).

Techniques: Expressing, Phospho-proteomics, Software