Glycogen synthase

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.

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 described^{57 (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).