Journal: Science Advances

Article Title: The skeletal muscle slow myosin heavy chain regulates mammalian metabolic homeostasis through the NRF2 pathway

doi: 10.1126/sciadv.aed2478

Figure Lengend Snippet: ( A to F ) Glucose tolerance test (GTT) on Pax +/+ , Myh7 fl/+ and Pax7 icre/+ , Myh7 Δ/fl 2-, 6-, and 12-month-old mice and their respective area under the curve ( n = 5 to 6). ( G to L ) Insulin tolerance test (ITT) of Pax +/+ , Myh7 fl/+ and Pax7 icre/+ , Myh7 Δ/fl mice at 2, 6, and 12 months and their respective area under the curve ( n = 5 to 6). ( M to Q ) Representative Western blots of P-Akt, Akt, and α-tubulin using protein lysates from the Gas muscle of Pax +/+ , Myh7 fl/+ and Pax7 icre/+ , Myh7 Δ/fl ( Myh7 KO ) mice at 2 and 6 months (M) and their densitometry (N to Q). α-Tubulin was used as a loading control ( n = 4). ( R to V ) Representative Western blots for GLUT4 and α-tubulin using protein lysates from the Gas muscle of Pax +/+ , Myh7 fl/+ , Pax3 cre/+ , Myh7 Δ/fl (P0), and Pax7 icre/+ , Myh7 Δ/fl mice at P0 and 2 and 6 months (R and S) and their densitometric quantification (T to V). α-Tubulin was used as a loading control ( n = 4 to 5). Data are presented as means ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The antibodies used for Western blotting were as follows: anti-mouse MyHC-IIa antibody (SC-71, Developmental Studies Hybridoma Bank) at 1:10 dilution; anti-mouse MyHC-IIb antibody (BFF3, Developmental Studies Hybridoma Bank) at 1:10; anti-mouse alpha-tubulin antibody (30021290, Cell Signaling Technology) at 1:10,000; anti-rabbit GAPDH antibody (PAB932Mu01, Cloud-Clone Corp) at 1:10,000; anti-rabbit Akt antibody (4641, Cell Signaling Technology) at 1:1000; anti-rabbit P-Akt S473 (4640, Cell Signaling Technology) at 1:1000; anti-rabbit Histone H3 antibody (Ab1741, Abcam) at 1:10,000; anti-rabbit troponin T1 antibody (NBP238855, Novus Biologicals) at 1:5000; anti-rabbit PGC-1α antibody (A12348, ABclonal; 66369, Proteintech; and 2178S, Cell Signaling Technology) at 1:2000, 1:2000, and 1:1000, respectively; anti-mouse total OXPHOS cocktail (ab110413, Abcam) at 1:1000; anti-rabbit DRP1 antibody (A21968, ABclonal) at 1:2000; anti-rabbit FIS1 antibody (A19666, ABclonal) at 1:2000; anti-rabbit mitofusin1 antibody (A9880, ABclonal) at 1:2000; anti-rabbit mitofusin2 antibody (A12771, ABclonal) at 1:2000; anti-rabbit GLUT4 (MA5-17176, Thermo Fisher Scientific) at 1:1000; anti-rabbit NRF2 antibody (A0674, ABclonal and 98020000, Proteintech) at 1:2000; anti-rabbit P-NRF2 antibody (AP1133, ABclonal) at 1:2000; anti-rabbit SOD1 antibody (A0274, ABclonal) at 1:2000; anti-rabbit Prdx1 antibody (A13524, ABclonal) at 1:2000; anti-rabbit Gpx1 antibody (A1110, ABclonal) at 1:1000; anti-rabbit catalase antibody (A11220, ABclonal) at 1:2000; anti-rabbit KEAP1 antibody (A1820, ABclonal) at 1:1000; anti-rabbit Tom20 antibody (HPA011582, Merck) at 1:2500, anti-rabbit VDAC1 antibody ( Ab154856 , Abcam) at 1:2500; anti-rabbit pDRP1 (S616) antibody (AP0849, ABclonal) at 1:2500; and anti-rabbit OPA1 antibody (A9833, ABclonal) at 1:2500.

Techniques: Western Blot, Control

Journal: Science Advances

Article Title: The skeletal muscle slow myosin heavy chain regulates mammalian metabolic homeostasis through the NRF2 pathway

doi: 10.1126/sciadv.aed2478

Figure Lengend Snippet: ( A ) MyHC-slow mediates normal oxidative phosphorylation levels and mitochondrial dynamics in the myofibers, maintaining normal blood glucose levels, insulin sensitivity, GLUT4 levels, mitochondrial function, NRF2 signaling, and MyHC proportions. ( B ) The absence of MyHC-slow results in reduced oxidative phosphorylation levels and increased mitochondrial fission in the myofibers, leading to increased ROS generation, muscle atrophy, altered MyHC proportions, and significant reduction in NRF2, GLUT4, and mitochondrial function, causing reduced insulin sensitivity and elevated blood glucose levels. FAPs, fibro-adipogenic progenitors. Created in BioRender. Mathew, S. (2026) https://BioRender.com/d6n81hh .

Article Snippet: The antibodies used for Western blotting were as follows: anti-mouse MyHC-IIa antibody (SC-71, Developmental Studies Hybridoma Bank) at 1:10 dilution; anti-mouse MyHC-IIb antibody (BFF3, Developmental Studies Hybridoma Bank) at 1:10; anti-mouse alpha-tubulin antibody (30021290, Cell Signaling Technology) at 1:10,000; anti-rabbit GAPDH antibody (PAB932Mu01, Cloud-Clone Corp) at 1:10,000; anti-rabbit Akt antibody (4641, Cell Signaling Technology) at 1:1000; anti-rabbit P-Akt S473 (4640, Cell Signaling Technology) at 1:1000; anti-rabbit Histone H3 antibody (Ab1741, Abcam) at 1:10,000; anti-rabbit troponin T1 antibody (NBP238855, Novus Biologicals) at 1:5000; anti-rabbit PGC-1α antibody (A12348, ABclonal; 66369, Proteintech; and 2178S, Cell Signaling Technology) at 1:2000, 1:2000, and 1:1000, respectively; anti-mouse total OXPHOS cocktail (ab110413, Abcam) at 1:1000; anti-rabbit DRP1 antibody (A21968, ABclonal) at 1:2000; anti-rabbit FIS1 antibody (A19666, ABclonal) at 1:2000; anti-rabbit mitofusin1 antibody (A9880, ABclonal) at 1:2000; anti-rabbit mitofusin2 antibody (A12771, ABclonal) at 1:2000; anti-rabbit GLUT4 (MA5-17176, Thermo Fisher Scientific) at 1:1000; anti-rabbit NRF2 antibody (A0674, ABclonal and 98020000, Proteintech) at 1:2000; anti-rabbit P-NRF2 antibody (AP1133, ABclonal) at 1:2000; anti-rabbit SOD1 antibody (A0274, ABclonal) at 1:2000; anti-rabbit Prdx1 antibody (A13524, ABclonal) at 1:2000; anti-rabbit Gpx1 antibody (A1110, ABclonal) at 1:1000; anti-rabbit catalase antibody (A11220, ABclonal) at 1:2000; anti-rabbit KEAP1 antibody (A1820, ABclonal) at 1:1000; anti-rabbit Tom20 antibody (HPA011582, Merck) at 1:2500, anti-rabbit VDAC1 antibody ( Ab154856 , Abcam) at 1:2500; anti-rabbit pDRP1 (S616) antibody (AP0849, ABclonal) at 1:2500; and anti-rabbit OPA1 antibody (A9833, ABclonal) at 1:2500.

Techniques: Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Analysis of multiple adipocyte PM proteomic datasets. A , schematic of the insulin treatments and PM isolation methods in different PM proteomic datasets. DS, datasets. LC-MS/MS, liquid chromatography-tandem mass spectrometry. GO-CC, Gene Ontology cellular component. B , UpSet plot showing the overlap of insulin-regulated ( p < 0.05) integral PM proteins across PM proteomic datasets with ≥3 biological replicates. C , Heatmap of 37 integral PM proteins identified by meta-analysis as significantly insulin-regulated and detected in ≥3 datasets. Values represent log 2 insulin-over-basal fold change (FOB); ND, not detected. D , insulin responsiveness of IRAP, GLUT4, TFR, KCC1, and PIT2 across nine datasets. E , Spearman correlation between IRAP, GLUT4, TFR, KCC1, and PIT2 mRNA expression in subcutaneous adipose tissue and metabolic clinical features. Figure generated from adiposetissue.org using data from ( , , , , , , , , , , , , , , , , , , , , ) (∗ pFDR < 0.05, ∗∗ pFDR < 0.01, ∗∗∗pFDR < 0.001). BMI, body mass index; circ, circulating; CRP, C-reactive protein; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment for insulin resistance; iso, isoproterenol; LDL, low-density lipoprotein; LEP, leptin; TG, triglycerides; WAT, white adipose tissue; WHR, waist-to-hip ratio. PM, plasma membrane; IRAP, insulin-regulated aminopeptidase; TFR, transferrin receptor; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4; pFDR, positive false discovery rate.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Expressing, Generated, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: KCC1 and PIT2 exhibit insulin dose-dependent PM recruitment. A , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 or KCC1-mStayGold were stimulated with different insulin doses (0.01–10 nM). PM recruitment was assessed by TIRF microscopy. Representative images for three independent experiments are presented (the scale bar represents 5 μm). Bas, basal condition (0.01 nM insulin). Ins, insulin stimulation. B , quantification of panel A . FOB, insulin-over-basal fold change. C and E , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( C ) or PIT2-HA ( E ) were stimulated with different insulin doses (0.01–100 nM). PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Representative images for two to three independent experiments are presented (the scale bar represents 20 μm). D , quantification of panels C and E . F , SGBS adipocytes stimulated with 0 or 10 nM insulin were fractionated to enrich PM proteins. Whole-cell lysates (WCL) and PM fractions were immunoblotted with the indicated antibodies. 14-3-3 and caveolin-1 (CAV1) served as loading controls for WCL and PM fractions, respectively. Representative blots from three independent experiments are shown. G , quantification of panel F , statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc ; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (n = 2–3 independent biological replicates). In ( B and D ), p values were compared to basal (0.01 nM insulin); in ( G ), p values were compared to GLUT4. PM, plasma membrane; TIRF, total internal reflection fluorescence; SGBS, Simpson–Golabi–Behmel syndrome; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Insulin-stimulated translocation of KCC1 and PIT2 to the PM requires PI3K-AKT signaling. A , schematic of the proximal insulin signaling pathway. IRS, insulin receptor substrates. PI3K, class I phosphoinositide 3-kinase. AKT, protein kinase B. B and D , 3T3-L1 adipocytes were pretreated for 10 min with 10 μM DMSO (vehicle; Ctrl), the PI3K inhibitor GDC-0941 (PI3Ki) ( B ), or the AKT inhibitor MK-2206 (AKTi) ( D ), then stimulated with 1 nM insulin. Cell lysates were immunoblotted with the indicated antibodies; 14-3-3 served as a loading control. Representative blots from three to four independent experiments are shown. C , quantification of panel B . E , quantification of panel D . F – H , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( F and H ) or KCC1-mStayGold ( G and H ) were treated as in ( B and D ), and PM recruitment was assessed by TIRF microscopy. I , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 or PIT2-HA were treated as in ( B and D ), and PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc ; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (n = 2–8 independent biological replicates). PM, plasma membrane; TIRF, total internal reflection fluorescence; PI3K, phosphoinositide 3-kinase; DMSO, dimethyl sulfoxide; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Translocation Assay, Control, Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Colocalization of KCC1/PIT2 with GLUT4 and TFR in unstimulated 3T3-L1 adipocytes. A and E , cells electroporated with either KCC1-mStayGold ( A ), or PIT2-HA ( E ) ( cyan ) were fixed, permeabilized, stained for nuclei ( gray ), GLUT4 ( yellow ), and TFR ( magenta ), and imaged using confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). B and F , quantification of panels A and E . C and G , cells were fixed, permeabilized, stained for nuclei ( gray ), GLUT4 ( yellow ), TFR ( magenta ), and KCC1 ( C ) or PIT2 ( G ) ( cyan ), and imaged using confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). D and H , quantification of panels C and G , quantitative analysis includes (i) percentage of each protein volume above threshold colocalized with GLUT4; (ii) percentage of GLUT4-positive volume colocalized with each protein; (iii) percentage of each protein volume colocalized with TFR; and (iv) percentage of TFR-positive volume colocalized with each protein. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test (n = 3 independent biological replicates). TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Impaired PIT2 and KCC1 translocation to the PM in insulin resistance. A – D , endogenous PM GLUT4 ( A and B ) and PM TFR ( C and D ) in 3T3-L1 adipocytes at basal and after acute 1 nM insulin, following 24-h pretreatment with 0 nM, 1 nM, or 10 nM insulin. Ctrl, control; CI_L, 1 nM chronic insulin; CI_H, 10 nM chronic insulin. Data are shown as raw PM intensity ( A and C ) or normalized to total GLUT4/TFR abundance ( B and D ), and expressed as percentage of the acutely insulin-stimulated control. E and F , 3T3L1 adipocytes electroporated with PIT2-HA were exposed to either 0, 1, or 10 nM insulin for 24 h, followed by stimulation with 1 nM insulin. PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Data are normalized to total PIT2-HA and expressed as percentage of the acutely insulin-stimulated control ( E ) or as the difference (%) between the acutely insulin-stimulated and basal PM intensities within each chronic-insulin condition ( F ). G and H , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( G ) or KCC1-mStayGold ( H ) were stimulated with 1 nM insulin after treatment with 0, one or 10 nM insulin for 24 h. PM recruitment was captured by TIRF microscopy. Data are shown as insulin-over-basal fold change (FOB). Statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. For ( F ), one-way ANOVA with Tukey’s post hoc was used (n = 4–6 independent biological replicates). PM, plasma membrane; TIRF, total internal reflection fluorescence; TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Translocation Assay, Control, Immunofluorescence, Staining, Confocal Microscopy, Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Chronic insulin alters the perinuclear localization of PIT2 and KCC1 under basal conditions. 3T3-L1 adipocytes were treated with 0, 1, or 10 nM insulin for 24 h, serum-starved (basal) for 2 to 3 h, then fixed, permeabilized, and stained for GLUT4 ( A and B ), PIT2 ( C and D ), KCC1 ( E and F ), or TFR ( G and H ). Cells were imaged by confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). Ctrl, control; CI_L, 1 nM chronic insulin; CI_H, 10 nM chronic insulin. Quantification shows the ratio of summed fluorescence intensity in the perinuclear region (PNR) to total cellular intensity. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test; ∗ p < 0.05, ∗∗ p < 0.01 (n = 3 independent biological replicates). TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Staining, Confocal Microscopy, Control, Fluorescence

Journal: Antioxidants

Article Title: PIN (Protein Inhibitor of Neuronal Nitric Oxide Synthase) Modulates Glucose Uptake Through NO-Dependent and Independent Mechanisms in Rat Muscle Cells

doi: 10.3390/antiox15040436

Figure Lengend Snippet: Effect of PIN overexpression on glucose uptake and GLUT4 translocation in L6 and primary muscle cells. ( A ) Western blotting analysis of PIN overexpression after transfection with PIN cDNA (PIN) or the empty vector (C) in L6 myocytes. ( B ) Effects of PIN overexpression on glucose uptake in L6 myocytes with or without 100 nM insulin. ( C ) Effects of PIN overexpression on glucose uptake in primary myotubes with or without 100 nM insulin. ( D ) Western blotting analysis of GLUT1 expression after transfection with PIN cDNA (PIN) or the empty vector (C) in L6 myocytes. ( E ) Western blotting analysis of GLUT4 expression after transfection with PIN cDNA (PIN) or the empty vector (C) in L6 myocytes. ( F ) Colorimetric immunoassay measuring GLUT4 translocation after PIN overexpression in L6 myocytes with or without 100 nM insulin. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: After saturation with PBS-0.4% BSA during 30 min, cells were incubated with an anti-GLUT4 antibody directed against 13 N-terminus extracellular amino acid of GLUT4 (GT42A, Alpha Diagnostic, San Antonio, TX, USA) overnight.

Techniques: Over Expression, Translocation Assay, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Antioxidants

Article Title: PIN (Protein Inhibitor of Neuronal Nitric Oxide Synthase) Modulates Glucose Uptake Through NO-Dependent and Independent Mechanisms in Rat Muscle Cells

doi: 10.3390/antiox15040436

Figure Lengend Snippet: Effect of nNOS blockade by L-NAME on glucose uptake and GLUT4 translocation in L6 and primary muscle cells. Effects of D-NAME ( A ) and L-NAME ( B ) on glucose uptake in L6 myocytes with or without 100 nM insulin. ( C ) Effects of L-NAME on glucose uptake in primary myotubes with or without 100 nM insulin. ( D ) Colorimetric immunoassay measuring GLUT4 translocation in L6 myocytes in the presence of 10 mM L-NAME with or without 100 nM insulin. ( E ) Effect of L-NAME on nNOS catalytic activity with or without 100 nM insulin. ( F ) Effects of SNP on glucose uptake in L6 myocytes with or without 100 nM insulin. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After saturation with PBS-0.4% BSA during 30 min, cells were incubated with an anti-GLUT4 antibody directed against 13 N-terminus extracellular amino acid of GLUT4 (GT42A, Alpha Diagnostic, San Antonio, TX, USA) overnight.

Techniques: Translocation Assay, Activity Assay

Journal: Antioxidants

Article Title: PIN (Protein Inhibitor of Neuronal Nitric Oxide Synthase) Modulates Glucose Uptake Through NO-Dependent and Independent Mechanisms in Rat Muscle Cells

doi: 10.3390/antiox15040436

Figure Lengend Snippet: Effects of PIN silencing and disruption with its interacting partners on glucose uptake in L6 muscle cells. ( A ) Western blotting analysis of PIN under-expression after transfection with PIN and control (C) siRNA. ( B ) Effects of PIN silencing on glucose uptake in L6 myocytes with or without 100 nM insulin. ( C ) Western blotting analysis of GLUT1 expression after transfection with PIN and control (C) siRNA. ( D ) Western blotting analysis of GLUT4 expression after transfection with PIN and control (C) siRNA. ( E ) Colorimetric immunoassay measuring GLUT4 translocation in L6 myocytes after PIN silencing in the presence or not of 100 nM insulin. ( F ) Western blotting analysis of nNOS expression in L6 myocytes after transfection with PIN or control (C) siRNA. ( G ) Effect of PIN silencing on nNOS catalytic activity in the absence of insulin. ( H ) Coimmunoprecipitation of nNOS with PIN in the presence of the control (peptide C) versus the inhibitory peptide (peptide I) in L6 myocytes. ( I ) Effects of the inhibitory peptide (peptide I) on glucose uptake in L6 myocytes with or without 100 nM insulin versus the irrelevant one (peptide C). ( J ) Effects of the inhibitory peptide (peptide I) on glucose uptake in primary myocytes from the insulin resistant Zucker fa/fa rat. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After saturation with PBS-0.4% BSA during 30 min, cells were incubated with an anti-GLUT4 antibody directed against 13 N-terminus extracellular amino acid of GLUT4 (GT42A, Alpha Diagnostic, San Antonio, TX, USA) overnight.

Techniques: Disruption, Western Blot, Expressing, Transfection, Control, Translocation Assay, Activity Assay