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anti phospho-akt (thr308)  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho-akt (thr308)
    Anti Phospho Akt (Thr308), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 5240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/phospho+akt+thr308/Phospho-Akt+(Thr308)+Antibody/pmc12765182-83-18-33
    Average 96 stars, based on 5240 article reviews
    anti phospho-akt (thr308) - by Bioz Stars, 2026-10
    96/100 stars

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    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Pluviatolide Attenuates Type I Hypersensitivity through Regulation of Mast Cell Activation.
    Article Snippet: Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA, USA). .. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% BSA in TBS-T. Membranes were incubated with primary antibodies against phospho-LYN (Tyr507), phospho-SYK (Tyr319), phospho-LAT (Tyr191), phospho-PLCγ1 (Tyr783), phospho-Akt (Thr308), phosphoERK1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), total Syk, LAT, AKT, ERK1/2, JNK, p38, NF-κB p65 purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and LYN, LAT, PLCγ1 purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). .. The secondary antibodies used were rabbit polyclonal antibody and mouse monoclonal antibody purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Article Title: Pluviatolide Attenuates Type I Hypersensitivity through Regulation of Mast Cell Activation
    Article Snippet: Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA, USA). .. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% BSA in TBS-T. Membranes were incubated with primary antibodies against phospho-LYN (Tyr507), phospho-SYK (Tyr319), phospho-LAT (Tyr191), phospho-PLCγ1 (Tyr783), phospho-Akt (Thr308), phospho-ERK1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), total Syk, LAT, AKT, ERK1/2, JNK, p38, NF-κB p65 purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and LYN, LAT, PLCγ1 purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). .. The secondary antibodies used were rabbit polyclonal antibody and mouse monoclonal antibody purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Incubation:

    Article Title: Pluviatolide Attenuates Type I Hypersensitivity through Regulation of Mast Cell Activation.
    Article Snippet: Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA, USA). .. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% BSA in TBS-T. Membranes were incubated with primary antibodies against phospho-LYN (Tyr507), phospho-SYK (Tyr319), phospho-LAT (Tyr191), phospho-PLCγ1 (Tyr783), phospho-Akt (Thr308), phosphoERK1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), total Syk, LAT, AKT, ERK1/2, JNK, p38, NF-κB p65 purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and LYN, LAT, PLCγ1 purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). .. The secondary antibodies used were rabbit polyclonal antibody and mouse monoclonal antibody purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Article Title: ATIC Knockdown Reduces B7-H3 Expression and Oncogenic Signaling in Upper Tract Urothelial Carcinoma Cells
    Article Snippet: .. Membranes were incubated overnight at 4°C with anti-ATIC (MA1-086, Invitrogen, Waltham, MA, USA), β-actin (#3700, Cell Signaling Technology, Danvers, MA, USA), α-tubulin (NB100-690, Novus Biologicals, Centennial, CO, USA), B7-H3 (#14058, Cell Signaling Technology), Prion Protein (A18058, ABclonalbio, New Taipei, Taiwan, ROC), RAC2 (A1139, ABclonalbio), NT5E (A25914, ABclonalbio), Fibronectin 1 (#26836, Cell Signaling Technology), Slug (NBP2-52570, Novus), Cyclin A2 (#4656, Cell Signaling Technology), Cyclin B1 (#4138, Cell Signaling Technology), p57 (NBP1-89917, Novus), phospho-mTOR (Ser2448) (SAB4504476, Sigma-Aldrich), mTOR (#2983, Cell Signaling Technology), phospho-AKT (Thr308) (#9275, Cell Signaling Technology), AKT (#9272, Cell Signaling Technology), phospho-p38 MAPK (Thr180/Tyr182) (#9211, Cell Signaling Technology), p38 MAPK (#9212, Cell Signaling Technology), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9101, Cell Signaling Technology), and p44/42 MAPK (Erk1/2) (#9102, Cell Signaling Technology). ..

    Article Title: Eight-week dietary methionine restriction does not impair resistance exercise-induced mTORC1 signalling activation in rats
    Article Snippet: .. After blocking, the membrane was washed in TBS-T and incubated overnight (4 °C) with primary antibody The following antibodies were used: p70S6K(#34475, CST), Phospho-p70S6K Thr389 (#9205, CST), rpS6 (#2217, CST), Phospho-rpS6 Ser240/244 (#2215, CST), 4E-BP1 (#9644, CST), Phospho-4E-BP1 Thr37/46 (#2885, CST), Puromycin (#MABE343, Merck Millipore, Burlington, MA, USA), Akt (#4691, CST), Phospho-Akt Thr308 (#13038, CST), Phospho-Akt Ser473 (#9271, CST), AMPKα (#2532, CST), Phospho-AMPKα Thr172 (#2537, CST), ULK1 (#8054, CST), Phospho-ULK1 Ser317 (#12753, CST), LC3B (#2775, CST), p62 (#PM045, Medical & Biological Laboratory, Nagoya, Japan), FoxO3a (#2497, CST), Phospho-FoxO3a Ser253 (#13129, CST), Fbx32 (#168372 Abcam, Cambridge, UK), MuRF-1 (#sc-398608, Santa Cruz Biotechnology, Dallas, TX, USA). .. The following day, the membrane was washed with TBS-T and the appropriate secondary antibody was added to TBS-T containing 1–3 % skim milk and incubated at room temperature for 1 h. The membrane was washed with TBS-T for 5 min and the bands were detected using Luminata Forte Western HRP Substrate (Millipore, CA, USA) with FUSION Chemiluminescence Imaging System (M&S Instruments, Osaka, Japan).

    Article Title: Pharmacological elevation of lactate alleviates sepsis via histone lactylation-induced IL-10 production.
    Article Snippet: Despite that lactate accumulation is deemed to be a marker of severe sepsis, lactate-driven histone lactylation induces transcription of homeostatic genes.. Thus, the biological roles of lactate in sepsis remain unknown.. Here, we report that amlexanox, an anti-inflammatory drug, improves survival, mitigates multiorgan dysfunction, and suppresses inflammatory infiltrates in endotoxemia and sepsis.

    Article Title: Pluviatolide Attenuates Type I Hypersensitivity through Regulation of Mast Cell Activation
    Article Snippet: Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA, USA). .. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% BSA in TBS-T. Membranes were incubated with primary antibodies against phospho-LYN (Tyr507), phospho-SYK (Tyr319), phospho-LAT (Tyr191), phospho-PLCγ1 (Tyr783), phospho-Akt (Thr308), phospho-ERK1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), total Syk, LAT, AKT, ERK1/2, JNK, p38, NF-κB p65 purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and LYN, LAT, PLCγ1 purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). .. The secondary antibodies used were rabbit polyclonal antibody and mouse monoclonal antibody purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Western Blot:

    Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters
    Article Snippet: .. Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036). .. Detection was carried out using an Odyssey CLx Imaging System (LI-COR Biosciences).

    Generated:

    Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters
    Article Snippet: .. Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036). .. Detection was carried out using an Odyssey CLx Imaging System (LI-COR Biosciences).

    other:

    Article Title: Nonlinear Bivariate Associations and Mononuclear Cell-Type-Specific Expression Level Differences in the STING Signalling Pathway.
    Article Snippet: Primary antibodies with specificities for STING (Thermo, cat# MA532768), phospho- STING ser 366 (Cell Signalling Technology: CST, cat# 40818), IRF3 (Abcam, cat# 68481), NLRP3 (Thermo, cat# MA532255), BDNF (Abcam, cat# ab108319), phospho- Akt thr308 (CST, cat# 2965), phospho- TBK1 ser172 (CST, cat# 5483), phospho- RelA ser536 (CST, cat# 3033), phospho- ULK1 ser757 (CST, cat# 14202), Traf6 (Abcam, cat# ab33915) and MyD88 (Abcam, cat# ab133739) were obtained from commercial sources.

    Blocking Assay:

    Article Title: Eight-week dietary methionine restriction does not impair resistance exercise-induced mTORC1 signalling activation in rats
    Article Snippet: .. After blocking, the membrane was washed in TBS-T and incubated overnight (4 °C) with primary antibody The following antibodies were used: p70S6K(#34475, CST), Phospho-p70S6K Thr389 (#9205, CST), rpS6 (#2217, CST), Phospho-rpS6 Ser240/244 (#2215, CST), 4E-BP1 (#9644, CST), Phospho-4E-BP1 Thr37/46 (#2885, CST), Puromycin (#MABE343, Merck Millipore, Burlington, MA, USA), Akt (#4691, CST), Phospho-Akt Thr308 (#13038, CST), Phospho-Akt Ser473 (#9271, CST), AMPKα (#2532, CST), Phospho-AMPKα Thr172 (#2537, CST), ULK1 (#8054, CST), Phospho-ULK1 Ser317 (#12753, CST), LC3B (#2775, CST), p62 (#PM045, Medical & Biological Laboratory, Nagoya, Japan), FoxO3a (#2497, CST), Phospho-FoxO3a Ser253 (#13129, CST), Fbx32 (#168372 Abcam, Cambridge, UK), MuRF-1 (#sc-398608, Santa Cruz Biotechnology, Dallas, TX, USA). .. The following day, the membrane was washed with TBS-T and the appropriate secondary antibody was added to TBS-T containing 1–3 % skim milk and incubated at room temperature for 1 h. The membrane was washed with TBS-T for 5 min and the bands were detected using Luminata Forte Western HRP Substrate (Millipore, CA, USA) with FUSION Chemiluminescence Imaging System (M&S Instruments, Osaka, Japan).

    Membrane:

    Article Title: Eight-week dietary methionine restriction does not impair resistance exercise-induced mTORC1 signalling activation in rats
    Article Snippet: .. After blocking, the membrane was washed in TBS-T and incubated overnight (4 °C) with primary antibody The following antibodies were used: p70S6K(#34475, CST), Phospho-p70S6K Thr389 (#9205, CST), rpS6 (#2217, CST), Phospho-rpS6 Ser240/244 (#2215, CST), 4E-BP1 (#9644, CST), Phospho-4E-BP1 Thr37/46 (#2885, CST), Puromycin (#MABE343, Merck Millipore, Burlington, MA, USA), Akt (#4691, CST), Phospho-Akt Thr308 (#13038, CST), Phospho-Akt Ser473 (#9271, CST), AMPKα (#2532, CST), Phospho-AMPKα Thr172 (#2537, CST), ULK1 (#8054, CST), Phospho-ULK1 Ser317 (#12753, CST), LC3B (#2775, CST), p62 (#PM045, Medical & Biological Laboratory, Nagoya, Japan), FoxO3a (#2497, CST), Phospho-FoxO3a Ser253 (#13129, CST), Fbx32 (#168372 Abcam, Cambridge, UK), MuRF-1 (#sc-398608, Santa Cruz Biotechnology, Dallas, TX, USA). .. The following day, the membrane was washed with TBS-T and the appropriate secondary antibody was added to TBS-T containing 1–3 % skim milk and incubated at room temperature for 1 h. The membrane was washed with TBS-T for 5 min and the bands were detected using Luminata Forte Western HRP Substrate (Millipore, CA, USA) with FUSION Chemiluminescence Imaging System (M&S Instruments, Osaka, Japan).



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    Cell Signaling Technology Inc primary antibodies against phospho akt thr308
    MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT <t>Thr308</t> levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .
    Primary Antibodies Against Phospho Akt Thr308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT <t>Thr308</t> levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .
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    MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT <t>Thr308</t> levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .
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    MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT Thr308 levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

    doi: 10.1084/jem.20251497

    Figure Lengend Snippet: MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT Thr308 levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .

    Article Snippet: Rabbit monoclonal anti-phospho-Akt (Thr308) , Cell Signaling Technology , Cat# 13038, RRID:AB_2629447.

    Techniques: Flow Cytometry, Expressing, Biomarker Discovery, Western Blot, Transfection, Staining, Migration, Knockdown, Phospho-proteomics, Comparison, Control