as160 tbc1d4  (Capra Science)

Journal: Redox Biology

Article Title: Revisiting insulin-stimulated hydrogen peroxide dynamics reveals a cytosolic reductive shift in skeletal muscle

doi: 10.1016/j.redox.2025.103607

Figure Lengend Snippet: Antibody information.

Article Snippet: TBC1D4 Thr642 , Cell Signaling Technology , 1:1000, 3 % skim milk , 4288.

Techniques:

Journal: Science Advances

Article Title: Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans

doi: 10.1126/sciadv.adq4461

Figure Lengend Snippet: ( A ) Schematic overview of the major signaling events modulating insulin-stimulated glucose uptake in human skeletal muscle. ( B ) Proximal insulin signaling, as determined by phosphorylation of Akt2 on Thr308 and Ser473 in whole-muscle homogenates. ( C and D ) Distal insulin signaling, as determined by phosphorylation of the Akt substrates GSK3β on Ser9 (C) and TBC1D4 on Thr642 (D) in whole-muscle homogenates. ( E ) GLUT4 translocation, as determined by GLUT4 protein abundance in plasma membrane protein fractions. In-gel stain-free technology was used as a loading control. Lipid, n = 8 (Pre-clamp) and n = 9 (End-clamp); Lipid + mtAO, n = 10. Representative blots ( n = 2 biological replicates from each muscle biopsy sample for each patient). ( F ) Purity of the isolated plasma membrane protein fractions (used to determine GLUT4 translocation), as determined by immunoblot analysis of actin (cytosolic protein marker) and Na + /K + -ATPase subunit α1 (plasma membrane protein marker) in plasma membrane homogenates (PM) as compared with the corresponding whole-muscle homogenates (WM). PM and WM samples were obtained by pooling a given volume of each individual sample. ( G ) Pearson’s correlation between mtAO-induced changes in plasma membrane GLUT4 and leg glucose uptake under insulin stimulation. n = 9. Data [(B) to (E)] presented as observed individual values with estimated means ±95% confidence limits. Linear mixed models were used to estimate within- and between-treatment differences at End-clamp [(B) to (E)]. *Different from Pre-clamp ( P < 0.05). n = 10, unless otherwise stated. Illustrations in (A) were created with BioRender.com .

Article Snippet: The following antibodies were used: Phospho-Akt (Thr308) (Cell Signaling, catalog no. 9275); Phospho-Akt (Ser473) (Cell Signaling, catalog no. 9271); Akt2 (Cell Signaling, catalog no. 3063); Phospho-GSK-3α/β (Ser21/9) (Cell Signaling, catalog no. 9331); GSK-3β (BD Transduction Laboratories, catalog no. 610202); Phospho-TBC1D4 (Thr642) (Cell Signaling, catalog no. 8881); TBC1D4 (Abcam, catalog no. ab189890); GLUT4 (Thermo Fisher Scientific, catalog no. PA1-1065); Na + /K + -adenosine triphosphatase (ATPase) subunit α1 (DSHB, catalog no. α6F); Actin (Sigma-Aldrich, catalog no. A2066); Myc-Tag (Cell Signaling, catalog no. 2272); PRDX2 (Abcam, catalog no. ab109367); PRDX3 (Abcam, catalog no. ab73349); PRDX-SO 2/3 (Abcam, catalog no. ab16830); 4-HNE (Alpha Diagnostics, catalog no. HNE11-s); Goat Anti-Rabbit Ig, Human ads-HRP (SouthernBiotech, catalog no. 4010-05); and Goat Anti-Mouse Immunoglobulins/HRP (Agilent Technologies, catalog no. P0447).

Techniques: Phospho-proteomics, Translocation Assay, Quantitative Proteomics, Clinical Proteomics, Membrane, Staining, Control, Isolation, Western Blot, Marker

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Effect of resveratrol on insulin action in primary myotubes from lean individuals and individuals with severe obesity

doi: 10.1152/ajpendo.00299.2023

Figure Lengend Snippet: Signatures on TBC1D4 phosphorylation in the absence or presence of 100 nM insulin in myotubes from lean and subjects with severe obesity in response to 24 h of resveratrol treatment (Resv). A : Thr642 phosphorylation. B : Ser704 phosphorylation. C : Ser341 phosphorylation. D : Ser588 phosphorylation. E : Ser318 phosphorylation. Following detecting significance for either group, treatment, and/or interaction effect using three-way ANOVA, distinguishing meaningful differences were performed using a two-tailed Student’s t test. There were main effects ( P < 0.05) for insulin for all measured sites of pTBC1D4s and for resveratrol for pThr642. There was a group effect with resveratrol for pThr642 and pSer704. Data are presented as means ± SE. n = 8/group (all females); * P < 0.05 for selected comparison. CTR, control.

Article Snippet: Primary antibodies were IRS1(Tyr632) (1:500, 09–433; Millipore, Billerica, MA); IRS1 protein (1:500, sc-559; Santa Cruz Biotechnology, Dallas, TX); Akt (Ser473) (1:1,000, 9271; Cell Signaling); Akt protein (1:1,000, 9272; Cell Signaling); AMP-activated protein kinase (AMPK) (Thr172) (1:1,000, 2531; Cell Signaling); AMPK protein (1:1,000, 2532; Cell Signaling); Akt-substrate at 160 kDa (AS160/TBC1D4) (Thr642) (1:1,000, ab59173; Abcam, Cambridge, MA); AS160/TBC1D4 (Ser588, Ser318, Ser341, Ser704) (1:1,000, customized by Capra Science, Sweden); AS160/TBC1D4 protein (1:1,000, 07-741; Millipore, Billerica, MA); beta actin (1:1,000, 926-42210; LI-COR Biosciences, Lincoln, NE); GLUT4 (1:1,000, sc-53566; Millipore); GLUT1 (1:1,000, Santa Cruz Biotechnology); peroxisome proliferator-activated receptor γ coactivator α (1:1,000, PGC1α) (ab106814; Abcam); SIRT1 (D739) (1:1,000, 2493; Cell Signaling); and citrate synthase (1:1,000, ab96600; Abcam).

Techniques: Two Tailed Test, Comparison, Control

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

Journal: bioRxiv

Article Title: GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells

doi: 10.1101/2023.06.06.543897

Figure Lengend Snippet: A. TBC1D4 mRNA expression in TBC1D4 KO cells. Two-sided paired Student’s T test on non-normalized data. Student’s 2 sample t-test. B. Fluorescence imaging for HA-GLUT4-GFP in differentiated SKM-CRISPR/CAS9 (control) and TBC1D4 KO SKM cells. Arrows note multinuclear cells. Scale bar 20µm. C. Assessment of Rab10 knockout by western blotting. D. Fluorescence imaging for HA-GLUT4-GFP in differentiated SKM-CRISPR/CAS9 (control) and Rab10 KO SKM cells. Arrows indicate multinuclear cells. Scale bar 20µm. E . GLUT4 rate constants measured in 4 independent experiments of control and Rab10 KO cells in unstimulated and PF739-stimulated conditions. F. Rab8a knockdown (KD) measured by western blotting. G. PM GLUT4 in unstimulated and PF739 stimulated control and Rab8a KD cells. Each symbol are data from independent experiments. One-way ANOVA p= 0.0002, p values FDR multiple comparison adjustment. H. Rab10 KD in SKM cells measured by western blotting. I. PM GLUT4 in unstimulated and PF739 stimulated control and Rab10 KD cells. Each symbol are data from independent experiments. PM GLUT4 in unstimulated and PF739 stimulated control and Rab8a KD cells. Each symbol are data from independent experiments. One-way ANOVA p < 0.0001, p values FDR multiple comparison adjustment.

Article Snippet: Primary antibodies used for Western blotting were against ß-tubulin (ab6046, Abcam), Rab10 (4262S, Cell Signaling Technology), Rab8A (610844, BD Biosciences), myosin heavy-chain (M4276, Millipore Sigma), myogenin (ab1835, Abcam), total acetyl-CoA carboxylase (ACC) (3676, Cell Signaling Technology), phospho-acetyl-CoA carboxylase (Ser79 ACC) (3661, Cell Signaling Technology), phospho T308-AKT (2965S, Cell Signaling Technology), total-AKT (9272S, Cell Signaling Technology), phospho-Thr642 TBC1D4 (8881S, Cell Signaling Technology), and total-TBC1D4 (07741, MilliporeSigma).

Techniques: Expressing, Fluorescence, Imaging, CRISPR, Control, Knock-Out, Western Blot, Knockdown, Comparison

Journal: bioRxiv

Article Title: GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells

doi: 10.1101/2023.06.06.543897

Figure Lengend Snippet: A. PM HA-GLUT4-GFP in day 3-differentiated SKM parental and TBC1D4 knockout (KO) cells stably expressing the HA-GLUT4-GFP, in basal (unstimulated) conditions. Each symbol is the mean value calculated from at least 40 cells in individual experiments (n=11). Data are presented as mean ± SEM. p, log Transformation Student’s 2 sample t-test 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 values ± SEM, n = 6. C. Exocytosis rate constants ± SEM determined from assays in panel B. Mean ± SEM, n=4. p, Welch’s 2 sample t-test. D. PM HA-GLUT4-GFP in day 3-differentiated SKM parental cells and SKM-Rab10 knockout (KO) cells stably expressing HA-GLUT4-GFP, treated with 3 µM PF739 for 60 minutes. Each symbol is the mean value of at least 40 cells per experiment (n=9). Data are presented as mean ± SEM normalized to the basal condition of parental cells. p values, One-way ANOVA with unequal variances p=0.0009, FDR multiple comparison adjustment. E. Exocytosis assay for HA-GLUT4-GFP under basal (unstimulated) conditions in day 3-differentiated SKM parental and Rab10-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 basal condition in each experiment. Data are mean values ± SEM, n = 4. F. Endocytosis assay for HA-GLUT4-GFP in day 3-differentiated SKM-CRISPR/CAS9 parental cells and Rab10-KO SKM cells stably expressing GLUT4 treated with or without 3 µM PF739 for 60 minutes. Data are a ratio of internal anti-HA (internalized pulse of GLUT4) to PM GLUT4. Data are normalized to the 6-minute time point of basal (unstimulated) condition in each experiment. Mean values ± SEM, n = 3.

Article Snippet: Primary antibodies used for Western blotting were against ß-tubulin (ab6046, Abcam), Rab10 (4262S, Cell Signaling Technology), Rab8A (610844, BD Biosciences), myosin heavy-chain (M4276, Millipore Sigma), myogenin (ab1835, Abcam), total acetyl-CoA carboxylase (ACC) (3676, Cell Signaling Technology), phospho-acetyl-CoA carboxylase (Ser79 ACC) (3661, Cell Signaling Technology), phospho T308-AKT (2965S, Cell Signaling Technology), total-AKT (9272S, Cell Signaling Technology), phospho-Thr642 TBC1D4 (8881S, Cell Signaling Technology), and total-TBC1D4 (07741, MilliporeSigma).

Techniques: Knock-Out, Stable Transfection, Expressing, Transformation Assay, CRISPR, Incubation, Comparison, Endocytosis Assay