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Glycogen synthase

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Glycogen synthase is a key enzyme involved in the synthesis of glycogen, the primary storage form of carbohydrates in the body. It catalyzes the addition of glucose units to the growing glycogen molecule.

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6 protocols using glycogen synthase

1

Glycogen Synthase Phosphorylation Analysis

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At the end of OGD-reoxygenation the cells were lysed in a cell lysis buffer (20 mM of Tris, 100 mM NaCl, 1 mM DiSodium-EDTA, 0.5% Triton-X) with protease and phosphatase inhibitors (1:100). Protein content of the samples was determined using Pierce 660 nm Protein assay reagent (Thermo Scientific). The samples were resolved on 10% SDS gel and transferred to a nitrocellulose membrane. The membranes were incubated overnight in primary antibodies against glycogen synthase (1:500; rabbit, Cell Signaling), Phospho-glycogen synthase (1:500; rabbit, Cell Signaling), and actin (1:1000; mouse, Santa Cruz) followed by secondary antibody (Jackson Immunoresearch, West Grove, PA). Using Biospectrum 500 UVP imaging system chemiluminescence signal was detected (Fig 6). The protein densities of glycogen synthase, Phospho-glycogen synthase were normalized to actin density.
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2

Western Blot Analysis of GSK3 and Related Proteins

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10-20 µg of protein was resolved by SDS-PAGE, transferred onto PVDF membranes (GSK3 phosphorylation, MilliporeSigma) or nitrocellulose membranes (GSK3/MARK inhbitors, Bio-Rad) and immunoblotted as previously described57 (link). Primary antibodies used were pS21/S9 GSK3α/β (9331S, Cell Signaling Technology), GSK3β (9315, Cell Signaling Technology), 14-3-3 (SC629, Santa Cruz Biotechnology), α-tubulin (T9026 Sigma Aldrich), pS641 glycogen synthase (47043, Cell Signaling Technology), glycogen synthase (3886, Cell Signaling Technology) and pS246/S259/S155 HDAC4/5/7 (3443, Cell Signaling Technology).
Membranes were incubated with the appropriate HRP-conjugated or Alexa Fluor-conjugated secondary antibodies for 1-2 h at room temperature. For GSK3 phosphorylation experiments protein bands were visualized by ECL (MilliporeSigma) on a LI-COR C-DiGit blot scanner (LI-COR Biosciences) or by 800-fluorescence intensity on a LI-COR Odyssey CLx imager. For GSK3/MARK inhibitor experiments protein bands were visualized using ECL (Thermo Fisher Scientific) or 647-fluorescence intensity on the Chemidoc MP (Bio-Rad).
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3

Comprehensive Western Blot Antibody Panel

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The following primary antibodies were used for western blotting at 1:1000 dilution: Ago2 (MBL CM004-3), Ago1 (MBL RN028PW), Glucokinase (Abcam ab88056), β-Actin (Sigma A1978), GAPDH (Abcam ab8245), α-Tubulin (Sigma, T6557), Glucose-6-phosphatase (Abcam ab83690), Glycogen synthase (Cell Signaling, #3893), Insulin receptor (Cell Signaling, #3025), Akt2 (Cell Signaling, #2964), phospho-Akt2 (Ser474) (Cell Signaling, #8599), FoxO1 (Cell Signaling, #2880), phospho-FoxO1 (Cell Signaling, #9464), Acetyl-CoA Carboxylase (Cell Signaling, #3676), phospho-Acetyl-CoA Carboxylase (Cell Signaling, #11818), Fatty Acid Synthase (Cell Signaling, #3180), AMPKα (Cell Signaling, #2532), and phospho-AMPKα (Cell Signaling, #2535). Image densitometry of 16-bit TIF images for all western blots was performed using ImageJ.
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4

Tissue Protein Lysate Immunodetection

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Tissue protein lysates were assayed with antibodies against Glycogen Synthase (Cell Signalling Technology, USA), GLUT1 and gp-91PHOX (Abcam, USA) GK and Srebpc1 (2A4) (Santa Cruz, USA). Protein levels were normalized to β-Actin (Abcam, USA). Secondary antibodies were horseradish peroxidise-coupled and ECL reagent (BioVision, USA) was used for detection. Quantification was performed using ImageGuage 4.0 (Fujifilm, Japan).
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5

Western Blot Analysis of Glucose Transporters

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Protein lysates were prepared by lysing cells in RIPA buffer containing protease inhibitor cocktail. The protein concentration was determined using the BCA protein assay (Thermo Scientific), and 30 μg of proteins were separated by SDS-PAGE and transferred to a 0.2 mm nitrocellulose membrane. The membranes were blocked by tris-buffered saline (TBS; 0.2 M Tris base, 1.5 M NaCl), 0.1% Tween-20, and 5% ECL blocking reagent (GE Healthcare) for one hour, then incubated with the primary antibody, GLUT1 (Abcam, ab15309), GLUT4 (Santa Cruz, sc-53566), Akt (Cell Signaling, #9272), P-Akt (Cell Signaling, #9271), heavy chain myosin (Abcam, ab124205) or glycogen synthase (Cell Signaling, #3893). Then, the membranes were incubated with HRP-conjugated secondary antibody (Abcam) diluted in TBS with 0.1% Tween-20. After the addition of the HRP substrate, the chemiluminescent signal was detected using a Gel and Blot Imaging System (Azure Biosystems). The same membrane was stripped and reused for detection of β-actin (Abcam, ab8229) or α-Tubulin (Sigma, #T6074) as a loading control, following the same protocol as above.
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6

Western Blot Analysis of Glucose Transporters

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Protein lysates were prepared by lysing cells in RIPA buffer containing protease inhibitor cocktail. The protein concentration was determined using the BCA protein assay (Thermo Scientific), and 30 μg of proteins were separated by SDS-PAGE and transferred to a 0.2 mm nitrocellulose membrane. The membranes were blocked by tris-buffered saline (TBS; 0.2 M Tris base, 1.5 M NaCl), 0.1% Tween-20, and 5% ECL blocking reagent (GE Healthcare) for one hour, then incubated with the primary antibody, GLUT1 (Abcam, ab15309), GLUT4 (Santa Cruz, sc-53566), Akt (Cell Signaling, #9272), P-Akt (Cell Signaling, #9271), heavy chain myosin (Abcam, ab124205) or glycogen synthase (Cell Signaling, #3893). Then, the membranes were incubated with HRP-conjugated secondary antibody (Abcam) diluted in TBS with 0.1% Tween-20. After the addition of the HRP substrate, the chemiluminescent signal was detected using a Gel and Blot Imaging System (Azure Biosystems). The same membrane was stripped and reused for detection of β-actin (Abcam, ab8229) or α-Tubulin (Sigma, #T6074) as a loading control, following the same protocol as above.
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