mtor substrates antibody sampler kit  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 intrinsic signaling induces the phosphorylation of the PI3K/Akt/mTORC1/STAT3 pathway and promotes RORγt binding on IL-17A regulatory elements (A) CCR6 + and CCR6 - CD4 T cells were sorted based on the expression of eGFP and stimulated with 100 ng/mL CCL20 at different time points (0, 5, 10, 15, 30, and 60 min). They were then immunoblotted with antibodies against various kinases in the downstream signaling pathways of Akt/mTOR/STAT3. (B) Sorted CCR6 + cells were rested overnight with or without rapamycin (50 ng/mL) and, the next day, stimulated with recombinant CCL20 (100 ng/mL) at two time points, 0 and 30 min. The phosphorylation status of STAT3 was analyzed by immunoblotting. Cyclophilin B and total STAT3 were used as loading controls. Blots are from 2 to 3 independent experiments. (C) The schematic presentation of IL-17A promoter (IL-17AP), IL-17F promoter (IL-17FP), conserved non-coding sequences (CNSs), and binding of RORγt. (D) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid vector containing the mouse IL-17 promoter (2 Kbp) and CNS5 enhancer elements. Transfected cells were stimulated with purified recombinant CCL20 (100 ng/mL) or TGF-β1 plus IL-6 at 37°C for 72 h. Then, cells were lysed, and luciferase activity was measured. The data shown are normalized to the Renilla luciferase activity. Each dot represents an independent experiment. Student’s t test. p -values are shown. (E) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid containing the IL-17 promoter (2 Kb) or IL-17 promoter (2 Kb) having a mutation in the RORγt binding site. Cells were cultured as given above, and luciferase activity was measured and plotted. The data shown are representative of one of the two independent experiments. The error bar represents ±SD. (F) Naive CD4 T cells were differentiated into the Th17 lineage condition (TGF-β + IL-6) in the presence or absence of CCL20 and with or without rapamycin (25 ng/mL) for 4 days. Expression of RORγt was analyzed after gating on CD4 + T cells. n = 3 experiments. (G) The schematic CCR6-CCL20 signaling pathways are shown. The p -value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: mTOR Substrates Antibody Sampler Kit , Cell Signaling Technology , Cat# 9862T; RRID: AB_10696885.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Protein-Protein interactions, Recombinant, Western Blot, Transfection, Plasmid Preparation, Purification, Luciferase, Activity Assay, Mutagenesis, Cell Culture, Comparison

Journal: Biomedicines

Article Title: E-Cadherin Regulates HIF1-α In Vitro in Induced 3D Spheroid Models of Human Breast Cancer Through Both mTOR and Notch1 Signaling

doi: 10.3390/biomedicines13122890

Figure Lengend Snippet: PI3K/AKT/mTOR signaling in induced spheroidgenesis. Western blot analysis shows mTOR activation and signaling during induced spheroidgenesis, which parallels increases in HIF-1α levels ( A ). Both mTOR signaling and HIF-1α levels are greater in E-cadherin-positive cell lines than -negative cell lines and are significantly inhibited by rapamycin, but Notch1 signaling is lower ( B , C ). Western blot also shows that PI3K/AKT signaling increases HIF-1α levels more in E-cadherin-positive cell lines than -negative cell lines ( D , E ). This inhibitor does not appreciably affect Notch1 signaling nor alterations in either PHD-2/Egln1 or VHL ( D , E ).

Article Snippet: Antibodies used included the following: Notch Activated Targets Antibody Sampler Kit, #68309, Cell Signaling Technology (CST), Danvers, MA, USA; Hypoxia Pathway Antibody, Sampler Kit, #15792, CST, which include antibodies to HIF-2α, VHL, hydroxy-HIF-1α, and PHD-2/Egln1; mTOR Substrates Antibody Sampler Kit, #9862, CST; and E-cadherin (G-10), #sc-8426, Santa Cruz Biotechnology, Santa Cruz, CA, USA; and Calpain 2 Large Subunit (M-type) (1:1000 dilution, #3195, (CST)).

Techniques: Western Blot, Activation Assay

Journal: Nature Aging

Article Title: Subcellular proteomics and iPSC modeling uncover reversible mechanisms of axonal pathology in Alzheimer’s disease

doi: 10.1038/s43587-025-00823-3

Figure Lengend Snippet: a , Workflow of the human iPSC-derived AD model. b , Image showing axonal spheroids (SMI312, gray) around amyloid deposits (thioflavinS, blue) and expressing ATG9A (red). c , Time-lapse imaging shows a spheroid forming (arrowhead) from a neurite (AAV9-hSyn-mCherry labeled) near Aβ deposits (gray) and enlarging over time. Lysosomes (AAV2-CMV-LAMP1-GFP labeled) accumulate within spheroids. d – h , Neuronal GCaMP8f imaging in the human iPSC AD model. d , Images of CAMKII-GCaMP8f-labeled neuronal processes with (upper) or without (lower) axonal spheroids and representative traces of calcium dynamics. y axis indicates Δ F / F , and dotted black lines indicate the calcium rise slope. Quantification of calcium rise time ( e ) and calcium rise speed ( f ). Each dot represents a neuronal process from three independent experiments (two-tailed Mann–Whitney test). g , Images showing that calcium decay time is slower in spheroids (pink asterisk) than in neuronal processes (blue asterisks). h , Quantification of calcium decay time in neuronal soma (blue), processes with (light pink) or without (light blue) spheroids and spheroids (pink). Each dot represents a neuronal process from three independent experiments (one-way ANOVA). i , mTOR signaling in iPSC-derived axonal spheroids (SMI312). j , Western blot showing that Torin1 treatment reduces mTOR downstream effectors phosphorylated 4E-BP1 and phosphorylated p70 S6K, whereas their total protein levels remain unchanged. k – r , Torin1 reduced axonal spheroids (SMI312) around Aβ deposits (thioflavin S). l – p , Pre-Aβ administration Torin1 treatment quantification: l , axon with spheroid percentage ( n = 3 in each group). Paired t -test two-tailed, P = 0.005. m , spheroid size (paired t -test two-tailed, P = 0.013, n = 4 per group). Dots represent experiments (20–30 ROIs). n , Axon number around plaques in each ROI (Torin1 n = 56; vehicle n = 55; unpaired t -test two-tailed, P = 0.880). o , Soma size. Dots represent neuronal somata (Torin1 n = 298, vehicle n = 316. Unpaired t -test two-tailed, P = 0.927; related to Extended Data Fig. ). p , Plaque size. Dots represent amyloid plaques (Torin1 n = 201, vehicle n = 253. Unpaired t -test two-tailed, P = 0.419). q , r , Post-Aβ administration Torin1 treatment (related to Extended Data Fig. ). Spheroid number normalized to axon density ( q ) and spheroid size ( r ) (Mann–Whitney test, two-tailed, n = 4 per group). Scale bar, 5 μm, except scale bar, 10 μm in g . e , f , h , l – r , Data presented as mean values ± s.e.m. See also Extended Data Figs. and and Supplementary Fig. . NS, not significant; ThioS, thioflavin S.

Article Snippet: To detect mTOR signaling substrates such as phosphor-4E-BP1 and p70S6K, an mTOR substrate antibody sampler kit (Cell Signaling Technology (CST), 9862) and anti-4E-BP1 antibody (CST, 9452) were used.

Techniques: Derivative Assay, Expressing, Imaging, Labeling, Two Tailed Test, MANN-WHITNEY, Western Blot

Journal: Nature Aging

Article Title: Subcellular proteomics and iPSC modeling uncover reversible mechanisms of axonal pathology in Alzheimer’s disease

doi: 10.1038/s43587-025-00823-3

Figure Lengend Snippet: a , Schematic of neuronal-specific conditional knockout of Mtor in heterozygous floxed mice. b , c , Images showing neuronal-specific Cre-mediated Mtor knockout: homozygous ( b ) and heterozygous ( c ), using AAV9-hSyn-Cre-2A-tdTomato or AAV PHPeB-hSyn-Cre-EGFP in Mtor -floxed mice, respectively. b , mTOR expression (gray) was absent in Cre-expressing neurons compared to adjacent NeuN-labeled neurons without Cre expression. c , mTOR expression was reduced in Cre-expressing neurons compared to neurons without Cre expression. Scale bar, 5 μm. d , Experimental design to study mTOR knockout effects on individual spheroids. e , Images showing AAV9-hSyn-Cre-2A-tdTomato sparsely labeling individual spheroids (red) within a spheroid halo (Lamp1, gray). f , Quantification of spheroid size. Dots represent animals (mTOR-flox-AD n = 5, 5×FAD n = 4. Unpaired t -test, two-tailed, P = 0.027). g , Using the same data in f , comparison of spheroid size distribution and visualization using a quantile–quantile (Q–Q) plot. Dashed lines indicate spheroid area at 10 µm 2 . 5×FAD mice have significantly more large spheroids (area > 10 µm 2 ) compared to mTOR-KO-AD (two-sample test for equality of proportions with continuity correction, two-tailed, P = 0.0004). h , Experimental design to assess the effect of Mtor knockout on spheroid halo size. i , Quantification of spheroid halo size. Dots represent animals ( n = 3; unpaired t -test, two-tailed, P = 0.041). j , Quantified by axonal spheroid halos (knockout group n = 66 and control group n = 109. Unpaired t -test, two-tailed, P < 0.0001). k , Quantification of neuronal soma size. Dots represent animals ( n = 3). Unpaired t -test, two-tailed, P = 0.90. l – o , Investigation of mTOR heterozygous knockout downstream signaling effectors. Immunofluorescence intensity of TFEB ( l ), LC3B ( m ), P-p70S6K Thr389 ( n ) and p70S6K ( o ). Littermates and sex were paired in l – o , paired t -test. Dots represent animals ( n = 3 in each group). p , RNAscope in 5×FAD mice cortices showing mRNA species (poly(A) probe, magenta) present in spheroids (NHS ester-labeled, yellow, and DAPI negative). NHS ester (yellow) labels the spheroid halo and amyloid plaques. Nuclei are labeled with DAPI (blue). Scale bar, 5 µm. q , Quantification of poly(A) probe fluorescence intensity versus negative control probe within spheroids in cortices of 5×FAD mice ( n = 3). Unpaired t -test, parametric, two-tailed, P = 0.001. r , Representative images showing puromycin labeling. Scale bar, 5 µm. s , Quantification of puromycin fluorescence intensity in axonal spheroids of 5×FAD mice ( n = 3). Unpaired t -test, parametric, two-tailed, P = 0.028. j , q , s , Data are presented as mean values ± s.e.m. See also Supplementary Figs. – . mo, months; NS, not significant.

Article Snippet: To detect mTOR signaling substrates such as phosphor-4E-BP1 and p70S6K, an mTOR substrate antibody sampler kit (Cell Signaling Technology (CST), 9862) and anti-4E-BP1 antibody (CST, 9452) were used.

Techniques: Knock-Out, Expressing, Labeling, Two Tailed Test, Comparison, Control, Immunofluorescence, RNAscope, Fluorescence, Negative Control

Journal: Frontiers in Oncology

Article Title: Fer governs mTORC1 regulating pathways and sustains viability of pancreatic ductal adenocarcinoma cells

doi: 10.3389/fonc.2024.1427029

Figure Lengend Snippet: E260 inhibits ERK1/2 signaling cascade and mTORC1 activation in PDAC cells. SU.86.86 and PANC-1 cells were left untreated or subjected to 2.5 µM E260 for 48 hr, in MEM supplied with 2mM L-glutamine. Cells were then harvested, and their lysates were resolved in SDS-PAGE and subjected to (A) anti-pospho-ERK1/2 (Thr.202/Tyr204), anti-ERK1/2, anti-phspho-TSC2 (Ser 664), anti-TSC, and anti-Tubulin. (B) anti-phospho-mTOR (Ser 2448), anti-mTOR, and anti-Tubulin, in a WB analysis. (C) Asparagine alleviates the death level evoked by E260 in PDAC cells. SU.86.86 cells were left untreated or treated with 2.5 µM E260, in the absence or presence of 4 mM asparagine, for 48 hr. The percentage of viable cells in each sample was determined using an automatic cell counter after the addition of Trypan blue to the sample. Data represent average values of three independent experiments that gave similar results. Standard deviations and P values are presented. (D) Lysates from each sample were resolved in SDS-PAGE and reacted with: anti-phospho-mTOR (Ser 2448), anti-mTOR (left panel), anti- phosphor-S6K (Thr389), anti-S6K (right panel), and anti-Tubulin, in a WB analysis. In each panel, one out of three independent experiments that gave similar results is presented.

Article Snippet: The following antibodies were used in the current work: affinity purified polyclonal anti-N-terminal Fer (anti-N-Fer, directed toward amino acids 1–189, of Fer), and anti-Fer -SH2 antibodies that were generated in our lab ( , )., anti-ATP5B mitochondrial ATP synthase beta subunit monoclonal antibody (Santa Cruz Biotechnology), anti-phospho-AMPK (Thr172) (Cell Signaling CST-#4188), anti-AMPKα1 (Santa Cruz Biotechnology sc-398861), anti -phospho-Raptor (Ser792) (Cell Signaling CST-2083P), anti-Raptor (Cell Signaling CST-9862T), anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phosphoTSC2 (Ser664), and anti-TSC2 (the mTOR Pathway Antibody Sampler Kit, Cell Signaling CST-9862T), anti-phospho-ERK1/2 (Thr202,Tyr204) (Sigma-Aldrich SAB4301578), anti-ERK1/2 (Abcam ab17942), anti-phospho-MEK1/2 (Ser218/222)- (Santa Cruz Biotechnology sc7995), anti-MEK1/2 (Santa Cruz Biotechnology sc-436), anti-NDUFA9 (Abcam-ab55521), anti-ATP5A (Abcam ab176569), anti-S6K (Abcam- ab186753), anti-phospho-S6K (Thr389) (Abcam ab32359), anti-actin (Santa Cruz Biotechnology sc-8432), and anti-α tubulin antibody (Santa Cruz Biotechnology sc-8035).

Techniques: Activation Assay, SDS Page

Journal: Frontiers in Oncology

Article Title: Fer governs mTORC1 regulating pathways and sustains viability of pancreatic ductal adenocarcinoma cells

doi: 10.3389/fonc.2024.1427029

Figure Lengend Snippet: Knockdown of Fer activates AMPK and downregulates ERK1/2 in PDAC cells. (A) SU.86.86. cells were transfected with control siRNA (siRNAc), or fer -targeting siRNA (siRNA fer ) and incubated in MEM supplied with 2 mM L-glutamine, for 72 hr. Cells were then harvested, and their lysates were resolved in SDS-PAGE and subjected to: anti-Fer (SH2), anti-pospho-ERK1/2 (Thr.202/Tyr204), anti-ERK1/2, anti-phspho-TSC2 (Ser 664), anti-TSC, anti-phospoh-AMPK (Thr172), anti-AMPK, anti-phospho-mTOR (Ser 2448), anti-mTOR, and anti-Tubulin, in a WB analysis. One out of three independent experiments that gave similar results is presented. (B) Summarizing scheme depicting the two regulatory pathways affected by E260 and converging to the downregulation of mTORC1 and mitochondrial function, in PDAC cells.

Article Snippet: The following antibodies were used in the current work: affinity purified polyclonal anti-N-terminal Fer (anti-N-Fer, directed toward amino acids 1–189, of Fer), and anti-Fer -SH2 antibodies that were generated in our lab ( , )., anti-ATP5B mitochondrial ATP synthase beta subunit monoclonal antibody (Santa Cruz Biotechnology), anti-phospho-AMPK (Thr172) (Cell Signaling CST-#4188), anti-AMPKα1 (Santa Cruz Biotechnology sc-398861), anti -phospho-Raptor (Ser792) (Cell Signaling CST-2083P), anti-Raptor (Cell Signaling CST-9862T), anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phosphoTSC2 (Ser664), and anti-TSC2 (the mTOR Pathway Antibody Sampler Kit, Cell Signaling CST-9862T), anti-phospho-ERK1/2 (Thr202,Tyr204) (Sigma-Aldrich SAB4301578), anti-ERK1/2 (Abcam ab17942), anti-phospho-MEK1/2 (Ser218/222)- (Santa Cruz Biotechnology sc7995), anti-MEK1/2 (Santa Cruz Biotechnology sc-436), anti-NDUFA9 (Abcam-ab55521), anti-ATP5A (Abcam ab176569), anti-S6K (Abcam- ab186753), anti-phospho-S6K (Thr389) (Abcam ab32359), anti-actin (Santa Cruz Biotechnology sc-8432), and anti-α tubulin antibody (Santa Cruz Biotechnology sc-8035).

Techniques: Knockdown, Transfection, Control, Incubation, SDS Page