Fresh surgical specimens were snap frozen in liquid nitrogen and stored in deep freezer. The normal and the tumor tissues were lysed in T-PER Tissue Protein Extraction Reagent(Pierce, Rockford, IL) containing proteinase inhibitors(CalBiochem, San Diego, CA). The extractions were collected and centrifuged at 12,000×g for 5 min. The protein concentrations were determined using the BCA Protein Assay (Pierce) according to the manufacturer's instructions. The following antibodies were used: anti-GSK-3β and anti-p-Ser9-GSK-3β (Cell Signaling Technology Inc., Beverly, MA), we also used β -actin as a loading control.
Anti p ser9 gsk 3β
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-Ser9-GSK-3β is a primary antibody that detects endogenous levels of GSK-3β only when phosphorylated at serine 9. This antibody is intended for use in western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti gsk3β, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti pser473 akt, by Cell Signaling Technology (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Evos m7000 imaging system, by Thermo Fisher Scientific (1 mentions)
Anti α actinin, by Abcam (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Solarbio (1 mentions)
Anti cd36, by Novus Biologicals (1 mentions)
Anti β mhc, by Abcam (1 mentions)
Anti mybpc3, by Santa Cruz Biotechnology (1 mentions)
Anti cpt1α, by Abcam (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
Anti ps473 akt, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
6 protocols using anti p ser9 gsk 3β
1
Protein Extraction and Quantification from Tumor and Normal Tissues
2
Western Blot Analysis of Phosphorylated Proteins
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The ipsilateral cortex tissue of the mouse brain was homogenized in prechilled buffer containing 50 mM Tris-HCl (pH 7.4), 2.0 mM EGTA, 2 mM Na3VO4, 50 mM NaF, 0.5 mM AEBSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 4 μg/ml pepstatin A. Protein concentrations of the homogenates were determined by using Pierce 660 nm Protein Assay kit (Thermo Fisher Scientific Inc.). The samples were resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto PVDF membrane (Millipore, Bedford, MA). After blocking with 5% fat-free milk, the blots were then probed with a primary antibody, such as VEGF (1 : 1000; Abcam, USA), anti-AKT (1 : 1000; Cell Signaling Technology, USA), anti-pSer473-AKT (1 : 1000; Cell Signaling Technology, USA), anti-pSer9-GSK3β (1 : 1000; Cell Signaling Technology, USA), anti-GSK3β (1 : 1000; Cell Signaling Technology, USA), or anti-GAPDH (1 : 2000; Sigma, USA),washed and then incubated with a corresponding HRP-conjugated secondary antibody. The protein-antibody complex was visualized by using the Pierce ECL Western Blotting Substrate (Thermo Scientific) and exposed to a HyBlot CL autoradiography film (Denville Scientific, Inc. Metuchen, NJ). Specific immunostaining was quantified by using the Multi Gauge software V3.0 (Fuji Photo Film Co. Ltd.).
3
Immunofluorescence Analysis of Apoptosis Markers
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Heart sections (4 μm) were fixed with 4% paraformaldehyde, blocked with 10% fetal bovine serum (FBS, #35-081-CV, Corning Inc., Corning, NY, USA), and permeabilized with Triton X-100. Anti-p-Ser9-GSK-3β (1:200, #93235, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved-capsase-1 (1:200, #PA5-99390, Invitrogen, Carlsbad, CA, USA), anti-GSDMD-NT (1:200, #PA5-115330, Invitrogen) and anti-ASC (1:200, #6741R-A5555, Bioss) antibodies were incubated overnight at 4°C, followed by secondary antibodies conjugated to Alexa Fluor 680 (1:500, #A10043, Invitrogen) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (#S2110, Solarbio, China). Anti-α-actinin was purchased from Abcam (Cambridge, MA, USA, 1:200, #ab68194). All slices were analyzed using the EVOS M7000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
4
Western Blot Analysis of Cardiac Proteins
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Protein concentration in heart tissue lysates was measured using the bicinchoninic acid method (Thermo Scientific, 23227, Waltham, MA, USA). Equivalent amounts of each protein extract were loaded into each well, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. Blots were probed with the following antibodies: anti-βMHC (Abcam, Cambridge, UK, ab50967), anti-MyBPC3 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-137237), anti-PPARα (Abcam, ab34509), anti-PPARβ/δ (Cell Signaling, Danvers, MA, USA, 2443S), anti-CD36 (Novus Biologicals, Minneapolis, MN, USA, NB400-144), anti-CPT1α (Abcam, ab1285568), anti-GLUT4 (Abcam, ab33780), anti-Akt (Cell Signaling, 9272S), anti-pS473 Akt (Cell Signaling, 9271S), anti-GSK3β (Cell Signaling, 5676S), anti-pSer9 GSK3β (Cell Signaling Technology, 9336S), and GAPDH (Proteintech, HRP-60004). Signals were visualized using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Image Lab Software version 6.0.1 (Bio-Rad) was used to quantify the differences in the fold induction of protein expression normalized to that of GAPDH.
5
Protein Kinase Signaling Pathways Analysis
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Western analyses were conducted with rabbit antisera or mouse monoclonal antibodies (16 (link)–21 (link)) using the following: anti-aPKC (Santa Cruz Biotechnology, Santa Cruz, CA); anti–p-Thr-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti–p-serine (Ser)-256-FOXO1 and anti-FOXO1 (Abnova, Walnut, CA); anti-Akt (mouse monoclonal antibodies), anti-p-Ser-473-Akt, anti–p-Ser-9-GSK3β, anti-GSK3β, anti–p-Ser-253-FOXO3a, anti-FOXO3a, anti–p-Ser-256-FOXO1, anti–p-Ser-193-FOXO4, anti-FOXO1, anti–p-Ser-2448-mammalian target of rapamycin (mTOR), anti-mTOR, anti–Aβ1–40/42 (5–10 kDa), and anti–amyloid precursor protein (APP; 120 kDa) (Cell Signaling Technology, Danvers, MA); and anti–p-Ser-202-tau and anti–p-Thr-231-tau (GeneTex, Irvine, CA). Samples from experimental groups were compared on the same blots and routinely checked with loading controls. Note that 75-kDa aPKC is largely PKC-λ in mouse brain and 98% homologous PKC-ι in brains of monkeys, humans, and other primates; PKC-ζ in brain exists largely as a 50-kDa moiety that, lacking a regulatory domain, is constitutively active and unresponsive to insulin; 50-kDa PKC-ζ (also called PKM-ζ) is produced by the operation of an intronal promoter and downstream transcription start site and is postulated to function in long-term memory potentiation (24 (link)); however, this idea has been challenged (25 (link),26 (link)).
6
Immunohistochemical Evaluation of GSK-3β and p-Ser9-GSK-3β
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Immunohistochemical staining was performed with the Dako Envision Plus System (Dako, Carpinteria, CA) according to the manufacturer's instructions. The primary antibodies were anti- GSK-3β and anti-p-Ser9-GSK-3β (Cell Signaling Technology Inc.,Beverly, MA, 1∶50). The tissues were evaluated as positive for GSK-3β and p-Ser9-GSK-3β staining when there were more than 10% of tumor cells demonstrating cytoplasmic and/or nucleus immunoreaction deposits. The sections were scored with a four-tier scale: 0 = negative (0–10%), 1 = weak signal (10–20%), 2 = intermediate signal (20–50%) and 3 = strong signal (> 50%). 0 and 1 were defined as low exprression, while 2 and 3 were defined as over-expression. All sections were scored independently by two observers who did not have any prior knowledge of the clinic-pathologic data. The concordance between scores from different sections of the same tumor was greater than 90%. All discrepancies in scoring were reviewed and a consensus was reached.
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