s473  (Cell Signaling Technology Inc)

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

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

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Taurine is the key small molecule in A1TP-HX-EVs that activated the AMPK/NRF2 pathway to regulate nucleus pulposus cell repair. (A) The LC-MS/MS analysis was used to detect the differential active small molecule components between placental HX-EVs and EVs. (B) The SMPDB enrichment analysis identified pathways related to small molecules that are up-expressed in HX-EVs compared to EVs. The metabolic pathways marked in red are related to ferroptosis inhibition and mitochondrial function. (C) Volcano plot of small molecule in HX-EVs versus EVs. |log2FC| > 0.5, FDR <0.05. (D) The content of taurine in placental MSC (pMSC), hypoxia-induced pMSC(HX-pMSC) and their derived EVs was detected by ELISA. n = 3. (E) Primary NPCs cells were induced with TBHP, and then treated with EVs, HX-EVs, and A1TP-HX-EVs for 24 h. The cell lysates were subjected to ELISA assay to detect taurine content. (F) Two shRNA lentiviruses were designed to knock down TAUT a key enzyme in taurine uptake in pMSC. (G) The content of taurine in TAUT-sh1-pMSC and TAUT-sh2-pMSC derived EVs (KD-HX-EVs) was detected by ELISA. n = 3. (H) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (I) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h, followed by immunofluorescent staining with anti- TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (J) A CDO1-overexpressing retrovirus was designed to overexpress CDO1 in pMSCs. (K) The content of taurine in CDO1-OE-pMSC derived EVs (OE-EVs) was detected by ELISA. n = 3. (L) Primary NPCs were induced with TBHP, and then treated with treated A1TP-EVs and A1TP-OE-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (M) Primary NPCs were induced with TBHP, and then treated with A1TP-EVs and A1TP-OE-EVs for 24 h, followed by immunofluorescent staining with anti-TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (N-O) Representative oxygen consumption traces of primary NPCs induced with TBHP and then treated with A1TP-HX-EVs, A1TP-KD-HX-EVs, or A1TP-OE-EVs for 24 h. Maximal respiration of NPCs were quantified. n = 3. All data are expressed as the mean ± SD. For E), I), M) and O), one‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. For D), G) and K), two‐tailed unpaired Student's t‐tests were used for statistical analysis. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001. ns, not significant.

Article Snippet: Primary antibodies included FTH1(4393S, Cell Signaling Technology), COL1A1(72026T, Cell Signaling Technology), COL2A1(sc-52658, Santa Cruz Biotechnology), MMP13(ab39012, Abcam), GPX4(30388-1-AP, Proteintech), ADGRG1(sc-390192, Santa Cruz Biotechnology), TAUT (sc-393036, Santa Cruz Biotechnology), TonEBP (sc-101098, Santa Cruz Biotechnology), CDO1 (12589-1-AP, Proteintech), AMPK(10929-2-AP, Proteintech), Phospho-AMPK (Thr172)(2535T, Cell Signaling Technology), SIRT1(8469T, Cell Signaling Technology), P-SIRT1(Ser47)(2314S, Cell Signaling Technology), PGC-1α(2178S, Cell Signaling Technology), Ac-lysine(sc-81623, Santa Cruz Biotechnology), NRF2(16396-1-AP, Proteintech), TFAM(22586-1-AP, Proteintech), NCOA4(66849S, Santa Cruz Biotechnology).

Techniques: Liquid Chromatography with Mass Spectroscopy, Inhibition, Derivative Assay, Enzyme-linked Immunosorbent Assay, shRNA, Knockdown, Staining, Comparison, Two Tailed Test