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).
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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