as160 t642  (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: The FASEB Journal

Article Title: Focal Adhesion Kinase Orchestrates GLUT4 Translocation and Glucose Uptake via Cytoskeletal Turnover in Primary Adipocytes

doi: 10.1096/fj.202402764rr

Figure Lengend Snippet: FIGURE 2 | (A) Representative western blots of insulin signaling targets in primary, inguinal mouse adipocytes after 1 h pre-treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin. (B) Corresponding quantifications of FAK Y397 (n = 3), IRS-1 Y612 (n = 3), PKB S473/T308 (n = 3), AS160 T642 (n = 3) and ERK1/2 T202/Y204 (n = 6) phosphorylation; data shown as mean ± SEM. Statistical analysis was performed using two-way repeated measures ANOVA with Bonferroni multiple comparison adjustment (α = 0.05). (C) Representative western blots of FAK Y397, PKB S473, AS160 T642, and ERK1/2 T202/Y204 in primary human subcutaneous adipocytes after 1 h pre-treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin; n = 1.

Article Snippet: Target Supplier Article No. RRID Dilution Notes AS160 Cell Signaling 2670 RRID:AB_2199375 1:1000 WB AS160 T642 Cell Signaling 4288 RRID:AB_10545274 1:500 WB FAK Cell Signaling 13 009 RRID:AB_2798086 1:1000 WB FAK Cell Signaling 3285 RRID:AB_2269034 1:50 IP FAK Y397 Cell Signaling 8556 RRID:AB_10891442 1:500 WB HSP90 BD Biosciences 610 418 RRID:AB_397798 1:2000 WB PKB S473 Cell Signaling 4060 RRID:AB_2315049 1:1000 WB PKB Cell Signaling 4691 RRID:AB_915783 1:1000 WB IRS- 1 Cell Signaling 3407 RRID:AB_2127860 1:250 WB IRS- 1 Y612 Thermo Fisher 44- 816G RRID:AB_1501247 1:500 WB ERK1/2 T202/Y204 Cell Signaling 4370 RRID:AB_2315112 1:1000 WB Phospho- PAK1 (T423)/PAK2 (T402) Cell Signaling 2601 RRID:AB_330220 1:1000 WB Arp2 (phospho T237 + T238) Abcam ab119766 RRID:AB_10900743 1:250 WB Phospho- Cofilin (S3) Cell Signaling 3313 RRID:AB_2080597 1:500 WB LM048 Integral Molecular CSB0148 RRID:AB_3106913 1:300 CM α- Tubulin Sigma- Aldrich CP06 RRID:AB_2617116 1:300 CM 15306860, 2025, 10, D ow nloaded from https://faseb.onlinelibrary.w iley.com /doi/10.1096/fj.202402764R R by N at Prov Indonesia, W iley O nline L ibrary on [22/05/2025].

Techniques: Western Blot, Incubation, Phospho-proteomics, Comparison

Journal: Journal of cell science

Article Title: Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

doi: 10.1242/jcs.261454

Figure Lengend Snippet: Fig. 2. TBC1D4 and Rab10 are required for AMPK regulation of GLUT4. (A) PM HA–GLUT4–GFP in day 3-differentiated SKM-CRISPR-Cas9 parental and TBC1D4 knockout (KO) cells stably expressing HA–GLUT4–GFP in basal (unstimulated) conditions. Each symbol is the mean calculated from at least 40 cells in individual experiments (n=16). Data are presented as mean±s.e.m. P-value calculated using a paired, two-tailed Student’s two-sample t-test on log-transformed data. (B) Exocytosis assay for HA–GLUT4–GFP under basal (unstimulated) conditions in day 3-differentiated SKM-CRISPR-Cas9 parental and TBC1D4 KO cells stably expressing HA–GLUT4–GFP. Data are cell-associated anti-HA as a function of incubation time in medium containing anti-HA antibody, normalized to the average of the 70, 120 and 150 min of basal condition in each experiment. Data are mean±s.e.m., n=4. Lines show the fit to a single exponential rise to a plateau. (C) Exocytosis rate constants (Ke), expressed as mean±s.e.m., determined from assays shown in B. n=4. P-value calculated using a two-tailed Welch’s two-sample t-test. (D) PM HA–GLUT4–GFP in day 3-differentiated SKM-CRISPR-Cas9 parental cells and Rab10 KO cells stably expressing HA–GLUT4–GFP, treated with or without 3 µM PF739 for 60 min. Each symbol is the mean value of at least 40 cells per experiment (n=8). Data are presented as mean±s.e.m., normalized to the basal condition of parental cells. P-value two-way ANOVA with unequal variances; P<0.0001; pairwise P-values in the figure have undergone FDR multiple comparison adjustment. (E) Exocytosis assay for HA–GLUT4–GFP under PF739-stimulated conditions in day 3-differentiated SKM parental and Rab10 (R10) KO cells stably expressing HA–GLUT4–GFP. Data are cell-associated anti-HA as a function of incubation time in medium containing anti-HA antibody, normalized to the average of 70, 120 and 150 min of control condition in each experiment. Data are mean±s.e.m., n=4. Lines show the fit to a single exponential rise to a plateau. (F) Endocytosis assay for HA–GLUT4–GFP in day 3-differentiated SKM- CRISPR-Cas9 parental cells and Rab10 KO cells stably expressing GLUT4 treated with or without 3 µM PF739 for 60 min. Data are a ratio of internal anti-HA (internalized pulse of GLUT4) to PM GLUT4. Data are normalized to the 6 min time point of basal (unstimulated) condition in each experiment. Mean±s.e.m., n=3. Lines show the fit to a straight line. A.U., arbitrary units.

Article Snippet: Primary antibodies used for western blotting were against β-tubulin (1:2000; ab6046, Abcam), Rab10 (1:500; 4262S, Cell Signaling Technology), Rab8A (1:1000; 610844, BD Biosciences), myosin heavy chain (1:1000; M4276, Millipore Sigma), myogenin (1:1000; ab1835, Abcam), total acetyl-CoA carboxylase (1:1000; 3676, Cell Signaling Technology), labelled-acetylCoA carboxylase (Ser79 ACC) (1:500; 3661, Cell Signaling Technology), labelled T308-AKT (1:500; 2965S, Cell Signaling Technology), total AKT (1:1000; 9272S, Cell Signaling Technology), labelled-T642 TBC1D4 (1:1000; 8881S, Cell Signaling Technology) and total TBC1D4 (1:1000; 07741, Millipore Sigma).

Techniques: CRISPR, Knock-Out, Stable Transfection, Expressing, Two Tailed Test, Transformation Assay, Incubation, Comparison, Control, Endocytosis Assay