primary antibody raised against glut4 (rabbit polyclonal; no. pa5-23052; lot wj3403509  (Thermo Fisher)

Journal: Nature Metabolism

Article Title: Skeletal muscle from TBC1D4 p.Arg684Ter variant carriers is severely insulin resistant but exhibits normal metabolic responses during exercise

doi: 10.1038/s42255-024-01153-1

Figure Lengend Snippet: a , Graphical representation of the EHC. b , GIR during the insulin clamp. c – e , GIR ( c ), endogenous glucose rate of appearance (RA) ( d ) and glucose rate of disappearance (RD) ( e ) at 120 min of the clamp. f , Plasma FAs during the clamp. g , Average leg glucose uptake during the last 40 min of the clamp. h , Targeted immunoblotting analyses of proteins associated with glucose metabolism, fat metabolism and the mitochondrial electron transport chain in skeletal muscle. i , Volcano plot showing proteome log 2 fold change (FC) (carriers/controls) plotted against –log 10 P value highlighting downregulated proteins TBC1D4, TBC1D1 and GLUT4 (SLC2A4). *Difference between TBC1D4 carriers and controls at the given time point. ‡Different from basal (time 0) within the same group. n = 5 ( b – g , i ). n = 7 ( h ) except for mTOR, p70S6K and ACC ( n = 5 in controls and TBC1D4 carriers) as well as Akt2, AMPKα2, CS, CD36 and FATP4 ( n = 6) in controls and GLUT1 ( n = 6) in TBC1D4 carriers. Data are means ± s.e.m. Data were analysed using a two-tailed paired Student’s t -test ( c – e , g ), a two-tailed non-paired Student’s t -test ( h ) as well as a two-way repeated ANOVA test (two-factor repeated) and two-tailed Student–Newman–Keuls post hoc analyses for multiple comparisons ( b , f ). Graphics in a created using BioRender.com . a.u., arbitrary units.

Article Snippet: For staining, four to six individual fibres were teased out from the fixed fibre bundles with fine forceps and blocked for 2 h in PBS containing 5% goat serum, 1% BSA and 0.04% saponin and then incubated overnight with primary antibody raised against GLUT4 (rabbit polyclonal; no. PA5-23052; lot WJ3403509, Thermo Scientific).

Techniques: Western Blot, Two Tailed Test

Journal: Nature Metabolism

Article Title: Skeletal muscle from TBC1D4 p.Arg684Ter variant carriers is severely insulin resistant but exhibits normal metabolic responses during exercise

doi: 10.1038/s42255-024-01153-1

Figure Lengend Snippet: a , Representative immunoblots for data related to figure panel 2 h. b , c , Distribution of Myosin Heavy Chains (MHC) ( b ) and regulatory metabolic ( c ) proteins in MHC-defined type 1 and type 2 skeletal muscle fiber bundles. d , Representative immunoblots. e , Quantification of GLUT4 imaging by confocal microscopy of isolated skeletal muscle fibers. n = 7 in Controls and n = 6 in TBC1D4 carriers ( b , c ). n = 15 fibers obtained from 3 subjects (4 to 6 fibers/subject) in each group ( e ). Data are means ± SEM. Data were analyzed using a two-tailed non-paired students t-test ( e ) as well as a two-way repeated ANOVA test (one factor repeated) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons ( b , c ). AU, arbitrary units. Scale bar in panel e is 10 µm.

Article Snippet: For staining, four to six individual fibres were teased out from the fixed fibre bundles with fine forceps and blocked for 2 h in PBS containing 5% goat serum, 1% BSA and 0.04% saponin and then incubated overnight with primary antibody raised against GLUT4 (rabbit polyclonal; no. PA5-23052; lot WJ3403509, Thermo Scientific).

Techniques: Western Blot, Imaging, Confocal Microscopy, Isolation, Two Tailed Test

Journal: The Journal of Physiology

Article Title: Rac1 governs exercise‐stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice

doi: 10.1113/JP272039

Figure Lengend Snippet: A, representative images showing cryosections of tibialis anterior muscle (cross sections) from basal and exercise‐stimulated Rac1 mKO and WT littermate mice stained with antibodies against GLUT4 (green) and α‐sarcoglycan (red). Scale bar = 50 μm. B, illustration of the quantification approach. Red arrows indicate the locations of the lines across and perpendicular to the plasma membrane of which 5 μm were analysed and quantified. C, quantification of the intensity of plasma membrane GLUT4 relative to α‐sarcoglycan in the basal and exercise‐stimulated (65% of individual maximal running speed, 20 min) state of WT and Rac1 mKO tibialis anterior muscle (n = 4–6). D, 2‐DG uptake in basal and exercise‐stimulated (65% of individual maximum running speed, 20 min) tibialis anterior muscle (n = 5 or 6). E, correlation between plasma membrane GLUT4 (GLUT4/α‐sarcoglycan) and 2‐DG uptake in basal and exercise‐stimulated tibialis anterior muscle. Included are only samples analysed for both parameters in the same muscle (n = 4 or 5). Significant difference between basal and exercise‐stimulated intensity of GLUT4/α‐sarcoglycan or 2‐DG uptake is indicated: * P < 0.05. Significant interaction between exercise and genotype is indicated: # P < 0.05. Values are the mean ± SEM.

Article Snippet: Cryosections were fixed for 30 min in ice‐cold 4% Zamboni buffer (4% paraformaldehyde, 0.15% picric acid, 0.1 m Sorensen's phosphate buffer, pH 7.3) and washed 3 × 10 min with phosphate‐buffered saline (PBS) and incubated for 2 h with primary antibodies raised against GLUT4 (rabbit polyclonal; #PA523052; Thermo Scientific, Waltham, MA, USA) and α‐sarcoglycan (mouse monoclonal, #IVD3(1)A9; Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Staining, Clinical Proteomics, Membrane