Pieces from the liver were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors. The protein concentration in the homogenates was determined using Bradford reagent [39 (link)] and 40 µg proteins were subjected to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The separated proteins were transferred to nitrocellulose membranes which were blocked using 5% skimmed milk in tris buffered saline/tween 20 (TBST). After blocking, the membranes were probed with antibodies against pAkt Ser473, Akt, pGSK-3β Ser9, GSK-3β, and β-actin (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. The blots were washed three times with TBST and incubated with the secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST and developed using enhanced chemiluminescence detection kit (BIO-RAD, Hercules, CA, USA). The developed blots were scanned, and the band intensity was quantified using ImageJ (version 1.32j, NIH, USA).
Gsk 3β
Manufactured by Novus Biologicals
Sourced in United States
GSK-3β is a serine/threonine protein kinase that plays a key role in the regulation of various cellular processes. It is involved in the Wnt signaling pathway and the regulation of glycogen metabolism, cell cycle, and apoptosis. GSK-3β is considered a potential therapeutic target for various diseases, including diabetes, neurological disorders, and cancer.
Automatically generated - may contain errors
Lab products found in correlation
Pgsk 3β ser9, by Novus Biologicals (3 mentions)
β actin, by Novus Biologicals (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence detection kit, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
Tnf α and il 6 elisa kits, by R&D Systems (1 mentions)
Agarose, by Merck Group (1 mentions)
Pyrogallol, by Merck Group (1 mentions)
Cuso4, by Merck Group (1 mentions)
P akt, by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
Caspase 3, by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Gapdh, by Abcam (1 mentions)
Sc 28338, by Santa Cruz Biotechnology (1 mentions)
5 protocols using gsk 3β
1
Western Blot Analysis of Liver Proteins
2
Western Blot Analysis of EMT Markers
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RIPA Buffer (Beyotime Biotechnology, China) containing 1% PMSF (Thermo, USA) was used to extract protein from cell and tissue samples, after which a BCA Kit (Beyotime) was employed to measure protein concentrations. Equal protein amounts from individual samples (cells: 30 ug; tissues: 60 ug) were subsequently separated via 10% SDS-PAGE and transferred to PVDF membranes (Millipore) that were then blocked for 2 h with 5% non-fat milk at room temperature, followed by overnight incubation with primary antibodies diluted in TBST containing 5% non-fat milk at 4°C. Blots were then incubated at room temperature with HRP-conjugated anti-rabbit or anti-mouse IgG (ZSBIO, China), after which a chemiluminescent imaging system was used for protein band detection. Primary antibodies included antibodies specific for the following, all of which were diluted at 1:1000 before use: ECM1 (Abcam, UK), E-cadherin (CST, USA), Vimentin (CST, USA), Snail (CST, USA), AKT (CST, USA), p-AKT (Ser473) (CST, USA), GSK3β (Novus, USA), p-GSK3β (Ser9) (Novus, USA), GAPDH (CST, USA). Image J was used for quantitative analyses.
3
Curcumin-Based Neuroprotection Assay
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CuSO4, CUR, carboxymethylcellulose (CMC), thiobarbituric acid, agarose, reduced glutathione (GSH), and pyrogallol were obtained from Sigma (St. Louis, MO, USA). Liposomal N-CUR was obtained from Lipolife (Essex, UK), and DFO was purchased from Novartis Pharma AG (Rotkreuz, Switzerland). TNF-α and IL-6 ELISA kits were supplied by R&D Systems (Minneapolis, MN, USA), and the NF-κB p65 ELISA kit was obtained from MyBiosource (San Diego, CA, USA). Antibodies against pAKT Ser473, AKT, pGSK-3β Ser9, GSK-3β, BDNF, and β-actin were supplied by Novus Biologicals (Centennial, CO, USA). Primers were obtained from Sigma (St. Louis, MO, USA). Other chemicals were supplied by standard manufacturers.
4
Western Blot Analysis of Signaling Pathways
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Protein lysates were prepared, and equal amounts of samples (20–30 µg for cell samples, 50–60 µg for tissue samples) were separated via 8%, 10%, or 12% SDS-PAGE based on target protein molecular weight values. PVDF membranes (Millipore, United States) were used to transfer the proteins, which were subsequently blocked with nonfat milk at 5% for 2 h. The blots were then probed overnight at 4°C with primary antibodies diluted in TBST. After three washes, blots were probed for 2 h with secondary antibodies (ZSBIO, China), and proteins were identified using a chemiluminescence imaging system (Tanon, China). Utilized antibodies were specific for E-cadherin (CST, United States), p-PI3K (Abcam, United Kingdom), PI3K (Abcam, United Kingdom), p-AKT (Abcam, United Kingdom), AKT (Abcam, United Kingdom), ECM1 (Abcam, United Kingdom), N-cadherin (CST, United States), p-GSK3β (Novus, United States), HRP-labeled Goat Anti-Rabbit IgG (ZSGBIO, China), GSK3β (Novus, United States), Bcl-2 (Affinity, China), Bax (Affinity, China), Caspase3 (Affinity, China) and GAPDH (CST, United States), and all were diluted 1:800–1:1000.
5
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed on the LV sections to evaluate the expression of BAX (LS-C353915, LSBio, Seattle, US), BCl-2 (BS-4563R, Bioss Inc., Boston, US), Caspase3 (SC-56046, Santa Cruz Biotechnology, CA, US), GSK-3β (NBP1-47470, Novus Biologicals, Manama, US), SGK1 (SC-28338, Santa Cruz Biotechnology, CA, US), P-AKT (Biotin, orb501663, Biorbyt, UK), P-ERK1/2 (orb10606, Biorbyt, UK), P-STAT3 (orb704362, Biorbyt, UK) proteins as previously described in detail elsewhere [7 (link)]. GAPDH (ab181602, Abcam, Cambridge, US) was used as the loading control. The intensity of each protein band was semi-quantified by the Image J image analysis system.
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