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Gsk 3

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GSK-3 is a laboratory equipment product. It is a protein kinase that plays a key role in various cellular processes. The core function of GSK-3 is to phosphorylate and regulate the activity of target proteins.

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Lab products found in correlation

Actin, by Santa Cruz Biotechnology (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Piaa 36, by Cell Signaling Technology (2 mentions) Rictor, by Cell Signaling Technology (2 mentions) Raptor, by Cell Signaling Technology (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions) Phospho rps6kb1, by Cell Signaling Technology (2 mentions) Phospho pkc, by Cell Signaling Technology (2 mentions) Phospho akt, by Cell Signaling Technology (2 mentions) Pharosfx plus, by Bio-Rad (2 mentions) Odyssey two color infrared imaging system, by LI COR (1 mentions) Ssea 1, by Merck Group (1 mentions) Flag m2 bead, by Merck Group (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Anti oct4, by Santa Cruz Biotechnology (1 mentions) P gsk3, by Cell Signaling Technology (1 mentions) Grp78 n 20, by Santa Cruz Biotechnology (1 mentions) Snai1, by Cell Signaling Technology (1 mentions) Cd133, by BioLegend (1 mentions) P akt, by Cell Signaling Technology (1 mentions)

6 protocols using gsk 3

1

Western Blot Analysis of Cell Signaling Proteins

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Proteins were separated using SDS-PAGE and transferred onto PVDF membranes. The antibodies were GFP 31 (link), 60 (link), FLAG (Sigma, F7425 and F3165), PiaA 36 (link), Tor (Cell Signaling, 2983), Rictor (Cell Signaling, 2114), Raptor (Cell Signaling, 2280), GAPDH (Thermo Fisher, MA5–15738), actin (Santa Cruz, Sc-1615), GSK-3 (Millipore, 05–412), phospho-RPS6KB1 (Cell Signaling, 9205), phospho-PKC (Cell Signaling, 2060), AKT (Cell Signaling, 9272) and phospho-AKT (serine 473) (Cell Signaling, 9271). Polyclonal rabbit antibodies to PKBA and PKBR1 were raised against the peptides, KNSDRKKRVNG and KKGNKNDETTP, respectively. Polyclonal rabbit antibodies to RacE and phospho-RacE (serine 192) were raised against the peptide REQQHPDPNSGKF and the phosphopeptide GMDKK(pS)QDGSS. Immunocomplexes were visualized using fluorescent-labeled secondary antibodies and detected using a Bio-Rad PharosFX Plus molecular imager. Images were analyzed using NIH ImageJ. The detailed information about the antibodies used in this study is provided in Supplementary Table 4.
Senoo H., Kamimura Y., Kimura R., Nakajima A., Sawai S., Sesaki H, & Iijima M. (2019). Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration. Nature cell biology, 21(7), 867-878.
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2

Protein Detection and Immunoprecipitation

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Cells were lysed with lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 12.5 mM b-glycerophosphate, 1.5 mM MgCl2, 2 mM ethylene glycol tetraacetic acid, 10 mM NaF, and 1 mM Na3VO4) containing protease inhibitors (Roche). Western blot was performed by standard procedures; primary antibodies used in this study: anti-Oct4 (Santa Cruz, sc-365509), anti-Rbm46 (sigma, HPA050601), beta-Actin (santa cruz, sc-47778), Pabpc1 (AVIVA, OAAB01699), GSK-3 (Millipore, 05–412), SSEA-1(Millipore, FCMAB117P). Proteins were visualized with an Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences) according to the manufacturer’s instructions. For Flag immunoprecipitaion, Flag M2 beads (Sigma, F1804) were utilized according to the manufacturer’s protocal.
Zhai L., Wang C., Chen Y., Zhou S, & Li L. (2017). Rbm46 regulates mouse embryonic stem cell differentiation by targeting β-Catenin mRNA for degradation. PLoS ONE, 12(2), e0172420.
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3

Antibody Sources for Stem Cell Research

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Antibody sources were as follows: GRP78 N20 and C20 [Santa Cruz Biotech (Santa Cruz, CA)]; OCT4, SOX9 and GSK3 [Millipore (Temecula, CA)]; CD133 and SCA1 [Biolegend (San Diego, CA)]; SNAI1, p-GSK3, AKT and p-AKT [Cell Signaling Technology (Danvers, MA)]. GRP78 C38 and C107 antibodies were produced in our laboratory (15 (link)).
Mo L., Bachelder R.E., Kennedy M., Chen P.H., Chi J.T., Berchuck A., Cianciolo G, & Pizzo S.V. (2015). Syngeneic murine ovarian cancer model reveals that ascites enriches for ovarian cancer stem-like cells expressing membrane GRP78. Molecular cancer therapeutics, 14(3), 747-756.
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4

Western Blot Analysis of Cell Signaling Proteins

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Proteins were separated using SDS-PAGE and transferred onto PVDF membranes. The antibodies were GFP 31 (link), 60 (link), FLAG (Sigma, F7425 and F3165), PiaA 36 (link), Tor (Cell Signaling, 2983), Rictor (Cell Signaling, 2114), Raptor (Cell Signaling, 2280), GAPDH (Thermo Fisher, MA5–15738), actin (Santa Cruz, Sc-1615), GSK-3 (Millipore, 05–412), phospho-RPS6KB1 (Cell Signaling, 9205), phospho-PKC (Cell Signaling, 2060), AKT (Cell Signaling, 9272) and phospho-AKT (serine 473) (Cell Signaling, 9271). Polyclonal rabbit antibodies to PKBA and PKBR1 were raised against the peptides, KNSDRKKRVNG and KKGNKNDETTP, respectively. Polyclonal rabbit antibodies to RacE and phospho-RacE (serine 192) were raised against the peptide REQQHPDPNSGKF and the phosphopeptide GMDKK(pS)QDGSS. Immunocomplexes were visualized using fluorescent-labeled secondary antibodies and detected using a Bio-Rad PharosFX Plus molecular imager. Images were analyzed using NIH ImageJ. The detailed information about the antibodies used in this study is provided in Supplementary Table 4.
Senoo H., Kamimura Y., Kimura R., Nakajima A., Sawai S., Sesaki H, & Iijima M. (2019). Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration. Nature cell biology, 21(7), 867-878.
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5

Quantitative Western Blot Analysis

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Whole-cell lysates were resolved by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with monoclonal antibodies against: GSK-3 (1:1000, Millipore Sigma, cat. no. 05-412), MITF (1:1000, SAB, cat. no. 33138), TYR (1:500, Abcam, cat. no. ab170905), TYRP1 (1:1000, Santa Cruz, cat. no. sc-166857), TYRP2 (1:1000, Santa Cruz, cat. no. sc-74439), and GAPDH (1:5000, Bioworld Technology, cat. no. AP0063). Bound antibodies were detected by incubation with secondary antibodies followed by enhanced chemiluminescence and visualized with a ChemiDoc XRS system. All the blots were accompanied by quantization and data were expressed as fold change of the densitometry value (normalized to the loading control) compared to the control using Image J software.
Jia Q., Tao L., Zhou Y., Song L., Wei Z., Lu T., Woodgett J.R, & Lu Y. (2022). Novel GSK-3 kinase inhibitor Pym-5 induces GSK-3β rather than GSK-3α-dependent melanogenesis in murine melanoma cells. Journal of dermatological science, 106(3).
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6

Protein Expression and Characterization

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Protein concentration, gel electrophoresis and transfer, and blotting were done as previously reported (72 (link)). In some instances, blots were stripped by incubation with 1× Western Reprobe (G-Biosciences, 786-119) for 45 minutes at 55°C followed by 3 washes with Tris-buffered saline with TWEEN-20 (CST, 9997) at room temperature. Antibodies used for Western blot studies were as follows: JAG2-N-terminal (CST, C23D2, 1:1000); JAG2-C-terminal (CST, C83A8, 1:1000); GSK3 (MilliporeSigma, 05-412, 1:2000); pGSK3 (MilliporeSigma, 0-413, 1:1000); TOP2A (Abcam, 109524, 1:10000); ATP1A1 (CST, 3010S, 1:2500); JAG1-C-terminal (BD, 612346, 1:1000); TUBA1A (MilliporeSigma, T6793, 1:2000); N-terminal β-catenin (CTNNB1, CST, 9581X, 1:1000); and C-terminal CTNNB1 (BD, 610154, 1:2000).
Reynolds S.D., Hill C.L., Alsudayri A., Lallier S.W., Wijeratne S., Tan Z.H., Chiang T, & Cormet-Boyaka E. (2022). Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease. JCI Insight, 7(15), e157380.
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