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akt substrate 160  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc akt substrate 160
    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and <t>AS160</t> phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 <t>S318,</t> and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.
    Akt Substrate 160, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+anti+phospho/Phospho-AS160+(Ser318)+Rabbit+mAb/pmc12816905-99-63-69
    Average 93 stars, based on 31 article reviews
    akt substrate 160 - by Bioz Stars, 2026-11
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    Images

    1) Product Images from "Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice"

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    Journal: Journal of Sport and Health Science

    doi: 10.1016/j.jshs.2025.101100

    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.
    Figure Legend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Techniques Used: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    Related Articles

    Western Blot:

    Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity
    Article Snippet: The BCA protein assay (Pierce, Thermo Fisher scientific) was conducted to determine protein concentration and following the manufacturer’s instructions, and for each sample, 20 μg of total protein was diluted using SDS sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol). .. The following primary antibodies were used for western blotting: goat anti-galectin-9 (AF2045; R&D Systems, Minneapolis, Minnesota) at 1:1,000 (vol/vol), rabbit anti-phospho-(Thr 423) Pak1 (#2601; Cell Signaling Technology) at 1:500 (vol/vol), rabbit anti-Pak1 (#2602; Cell Signaling Technology) at 1:500 (vol/vol), rabbit anti-GAPDH (#2118; Cell Signaling Technology) at 1:500 (vol/vol), mouse anti-pMLC (#3675; Cell Signaling Technology) at 1:500 (vol/vol), rabbit anti-mDia (#DP4471; BD/ECM Biosciences) at 1:500 (vol/vol), and rat anti-tubulin (Novus Biological) at 1:2,000 (vol/vol). .. The following secondary antibodies were used: donkey anti-goat IRDye 680 (920-32224; LI-COR), donkey anti-rabbit IRDye 800 (926-32213; LI-COR), donkey anti-rabbit IRDye 680 (926-68073; LI-COR), goat anti-rabbit IRDye 800 (926-32211; LI-COR), goat anti-rat IRDye 680 (A21096; Invitrogen), donkey anti-mouse IRDye 680 (926-68072; LI-COR).

    other:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling.
    Article Snippet: The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).

    Blocking Assay:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
    Article Snippet: The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). .. After blocking with Tris-buffered saline (TBS) with 5% skim milk (w/v) for 1 h, the membranes were incubated with rabbit anti-HMGB1, mouse anti-Myc (Invitrogen, 13–2500), rabbit anti-MD2, rabbit anti-phospho–NF–κB p65 (Ser536) (Cell Signaling, 3033), rabbit anti–NF–κB p65 (Cell Signaling, 8242), and rabbit anti-actin (Cell Signaling, 4967) antibodies overnight at 4°C or for 2 h at room temperature (RT). .. The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).

    Saline:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
    Article Snippet: The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). .. After blocking with Tris-buffered saline (TBS) with 5% skim milk (w/v) for 1 h, the membranes were incubated with rabbit anti-HMGB1, mouse anti-Myc (Invitrogen, 13–2500), rabbit anti-MD2, rabbit anti-phospho–NF–κB p65 (Ser536) (Cell Signaling, 3033), rabbit anti–NF–κB p65 (Cell Signaling, 8242), and rabbit anti-actin (Cell Signaling, 4967) antibodies overnight at 4°C or for 2 h at room temperature (RT). .. The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).

    Transcranial Magnetic Stimulation:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
    Article Snippet: The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). .. After blocking with Tris-buffered saline (TBS) with 5% skim milk (w/v) for 1 h, the membranes were incubated with rabbit anti-HMGB1, mouse anti-Myc (Invitrogen, 13–2500), rabbit anti-MD2, rabbit anti-phospho–NF–κB p65 (Ser536) (Cell Signaling, 3033), rabbit anti–NF–κB p65 (Cell Signaling, 8242), and rabbit anti-actin (Cell Signaling, 4967) antibodies overnight at 4°C or for 2 h at room temperature (RT). .. The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).

    Incubation:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
    Article Snippet: The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). .. After blocking with Tris-buffered saline (TBS) with 5% skim milk (w/v) for 1 h, the membranes were incubated with rabbit anti-HMGB1, mouse anti-Myc (Invitrogen, 13–2500), rabbit anti-MD2, rabbit anti-phospho–NF–κB p65 (Ser536) (Cell Signaling, 3033), rabbit anti–NF–κB p65 (Cell Signaling, 8242), and rabbit anti-actin (Cell Signaling, 4967) antibodies overnight at 4°C or for 2 h at room temperature (RT). .. The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).

    Cell-Signaling:

    Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
    Article Snippet: The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). .. After blocking with Tris-buffered saline (TBS) with 5% skim milk (w/v) for 1 h, the membranes were incubated with rabbit anti-HMGB1, mouse anti-Myc (Invitrogen, 13–2500), rabbit anti-MD2, rabbit anti-phospho–NF–κB p65 (Ser536) (Cell Signaling, 3033), rabbit anti–NF–κB p65 (Cell Signaling, 8242), and rabbit anti-actin (Cell Signaling, 4967) antibodies overnight at 4°C or for 2 h at room temperature (RT). .. The membranes were then washed with TBS containing 0.1% tween 20 (TBST) and incubated at RT for 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson laboratories, 111-035-003 and 111-035-003).



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    IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
    Rabbit Anti Phospho Stat1 Y701, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi3k p85α  (ABclonal Biotechnology)
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    IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
    Pi3k P85α, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho acc1 s79  (ABclonal Biotechnology)
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    IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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    Image Search Results


    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Journal: Journal of Sport and Health Science

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    doi: 10.1016/j.jshs.2025.101100

    Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

    Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Journal: Journal of Sport and Health Science

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    doi: 10.1016/j.jshs.2025.101100

    Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

    Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    ( A ) Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice 5 months after intranasal inoculation with Adeno-Cre (left). Tumor incidence, size, and burden (tumor area/total lung area × 100) were quantified (second to fourth panels, respectively). Data presented as means and SD. Statistical analysis was performed by one-way ANOVA and Student’s t test. ( B ) Representative IHC staining of CASTOR1 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice. ( C ) Representative IHC staining images of Ki67 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left) and quantification of Ki67-positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( D and E ) Representative IHC staining images of p-4EBP1 (D) and p-S6 (E) of lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panels) and quantification of positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( F ) Immunoblotting analysis of mTORC1 downstream targets p-S6K and p-4EBP1, KRAS downstream target ERK and AKT, and their phosphorylated forms in normal lung tissues from KRAS WT ;C1 WT and KRAS WT ;C1 KO mice and tumors from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO mice (left panels). Three samples per group were analyzed and quantified (right panels). Statistical analyses were performed using nested-ANOVA followed by Tukey’s post hoc test for multiple comparisons. ( G ) Representative IHC staining images of p-ERK in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (top panels) and quantification of positive cells (bottom). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test or Student’s t test, as appropriate. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

    Journal: Science Advances

    Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

    doi: 10.1126/sciadv.aeb9836

    Figure Lengend Snippet: ( A ) Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice 5 months after intranasal inoculation with Adeno-Cre (left). Tumor incidence, size, and burden (tumor area/total lung area × 100) were quantified (second to fourth panels, respectively). Data presented as means and SD. Statistical analysis was performed by one-way ANOVA and Student’s t test. ( B ) Representative IHC staining of CASTOR1 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice. ( C ) Representative IHC staining images of Ki67 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left) and quantification of Ki67-positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( D and E ) Representative IHC staining images of p-4EBP1 (D) and p-S6 (E) of lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panels) and quantification of positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( F ) Immunoblotting analysis of mTORC1 downstream targets p-S6K and p-4EBP1, KRAS downstream target ERK and AKT, and their phosphorylated forms in normal lung tissues from KRAS WT ;C1 WT and KRAS WT ;C1 KO mice and tumors from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO mice (left panels). Three samples per group were analyzed and quantified (right panels). Statistical analyses were performed using nested-ANOVA followed by Tukey’s post hoc test for multiple comparisons. ( G ) Representative IHC staining images of p-ERK in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (top panels) and quantification of positive cells (bottom). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test or Student’s t test, as appropriate. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

    Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

    Techniques: Analysis, Immunohistochemistry, Western Blot

    ( A to C ) CASTOR1 loss enhances resistance to KRAS G12D -specific inhibitor MRTX1133 in tumors. Mice were intranasally inoculated with Adeno-Cre for 4 weeks and then treated with MRTX1133 or vehicle for 4 weeks. Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice were shown (A). Tumor size (B) and burden (tumor area/total lung area × 100) (C) were quantified. Results presented as mean and SD. Statistical analysis was performed by nested ANOVA, followed by Tukey’s post hoc test for multiple comparisons. ( D ) KRAS G12D ;C1 WT tumor organoids had heterogeneous response to MRTX1133. Representative pictures of nine KRAS G12D ;C1 WT organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( E ) Immunoblotting analysis of KRAS G12D protein, mTORC1 downstream targets S6K and 4EBP1, KRAS downstream target ERK, and their phosphorylated forms in KRAS G12D ;C1 WT tumor organoids. ( F ) Regression analysis of KRAS G12D , S6K, 4EBP1, and ERK and their phosphorylated forms versus organoid viability, showing the strongest correlation with p-S6K, followed by p-4EBP1. ( G ) KRAS G12D ;C1 KO tumor organoids were resistant to MRTX1133. Representative pictures of four KRAS G12D ;C1 KO organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( H ) Comparison of sensitivity to MRTX1133 between organoids derived from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO tumors. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

    Journal: Science Advances

    Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

    doi: 10.1126/sciadv.aeb9836

    Figure Lengend Snippet: ( A to C ) CASTOR1 loss enhances resistance to KRAS G12D -specific inhibitor MRTX1133 in tumors. Mice were intranasally inoculated with Adeno-Cre for 4 weeks and then treated with MRTX1133 or vehicle for 4 weeks. Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice were shown (A). Tumor size (B) and burden (tumor area/total lung area × 100) (C) were quantified. Results presented as mean and SD. Statistical analysis was performed by nested ANOVA, followed by Tukey’s post hoc test for multiple comparisons. ( D ) KRAS G12D ;C1 WT tumor organoids had heterogeneous response to MRTX1133. Representative pictures of nine KRAS G12D ;C1 WT organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( E ) Immunoblotting analysis of KRAS G12D protein, mTORC1 downstream targets S6K and 4EBP1, KRAS downstream target ERK, and their phosphorylated forms in KRAS G12D ;C1 WT tumor organoids. ( F ) Regression analysis of KRAS G12D , S6K, 4EBP1, and ERK and their phosphorylated forms versus organoid viability, showing the strongest correlation with p-S6K, followed by p-4EBP1. ( G ) KRAS G12D ;C1 KO tumor organoids were resistant to MRTX1133. Representative pictures of four KRAS G12D ;C1 KO organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( H ) Comparison of sensitivity to MRTX1133 between organoids derived from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO tumors. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

    Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

    Techniques: Analysis, Organoids, Western Blot, Comparison, Sensitivity, Derivative Assay

    ( A ) Knockdown of CASTOR1 enhanced cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following siRNA knockdown of CASTOR1 . ( B ) Overexpression of CASTOR1 inhibited cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following the overexpression of CASTOR1 . ( C ) Knockdown of CASTOR1 enhanced resistance to mTORC1 inhibitor Torin1 in human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines treated with Torin1 following siRNA knockdown of CASTOR1 . ( D ) Overexpression of CASTOR1 sensitized human lung cancer cell lines to mTORC1 inhibitors Torin1 and rapamycin. Clonogenic assay and immunoblotting of human lung cancer cell lines with mTOR inhibitors Torin1 and rapamycin following the overexpression of CASTOR1 . Rapamycin was used at 20 nM, while Torin1 was used at 0.2 μM.

    Journal: Science Advances

    Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

    doi: 10.1126/sciadv.aeb9836

    Figure Lengend Snippet: ( A ) Knockdown of CASTOR1 enhanced cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following siRNA knockdown of CASTOR1 . ( B ) Overexpression of CASTOR1 inhibited cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following the overexpression of CASTOR1 . ( C ) Knockdown of CASTOR1 enhanced resistance to mTORC1 inhibitor Torin1 in human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines treated with Torin1 following siRNA knockdown of CASTOR1 . ( D ) Overexpression of CASTOR1 sensitized human lung cancer cell lines to mTORC1 inhibitors Torin1 and rapamycin. Clonogenic assay and immunoblotting of human lung cancer cell lines with mTOR inhibitors Torin1 and rapamycin following the overexpression of CASTOR1 . Rapamycin was used at 20 nM, while Torin1 was used at 0.2 μM.

    Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

    Techniques: Knockdown, Clonogenic Assay, Western Blot, Over Expression

    IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Journal: Immunity, Inflammation and Disease

    Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

    doi: 10.1002/iid3.70564

    Figure Lengend Snippet: IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Article Snippet: Primary antibodies for immunofluorescence and Western blotting: EBI3 (12371‐1‐AP), IL‐12A (85754‐2‐RR), platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) (11265‐1‐AP), eNOS (27120‐1‐AP), and β‐tubulin (66240‐1‐Ig) from Proteintech (USA); p‐eNOS (Ser1177, #9571), p‐cRAF (#9427), p‐MEK1/2 (#9154), and p‐STAT3 (#9145) from Cell Signaling Technology (USA); cRAF (A19638), MEK1/2 (A4868), p‐ERK1/2 (AP0974), ERK1/2 (A4782), and STAT3 (A1192) from ABclonal Technology (China).

    Techniques: Phospho-proteomics, Western Blot, Analysis, Fluorescence