Cell extracts containing 40 μg of total protein were separated by electrophoresis on sodium dodecyl sulphate polyacrylamide gels, and subsequently transferred to nitrocellulose membranes. After transfer, the membranes were blocked with PBS containing 5% non‐fat milk for 1 hour at room temperature, and then incubated at 4°C overnight with primary antibodies against Total AKT (T‐AKT, Boster, Wuhan, China), Phospho‐AKT (P‐AKT; Cell Signaling Technology), T‐GSK‐3β (Cell Signaling Technology), Phospho‐GSK‐3β (Cell Signaling Technology), β‐catenin (Cell Signaling Technology), Cyclin D1 (Abcam, Cambridge, MA, USA), Runt‐related transcription factor 2 (RUNX2; Boster), Bone morphogenic protein‐2 (BMP2; Boster) and Bone sialoprotein (BSP; Boster). Antibody against β‐actin (Sigma‐Aldrich) was used as normalizing control. The membranes were subsequently incubated with the secondary antibodies for 1 hour at room temperature. The results were analysed using an Odyssey 2‐colour infrared laser imaging system (LI‐COR Biosciences, Lincoln, NE, USA). Relative density of labelled protein band was analysed by Image‐ProPlus 5.0 software (Media Cybernetics Inc, Rockville, MD, USA)
T gsk3β
Manufactured by Cell Signaling Technology
Sourced in United States
T-GSK3β is a phospho-specific antibody that recognizes the phosphorylated form of glycogen synthase kinase-3 beta (GSK3β) at threonine 217. GSK3β is a serine/threonine protein kinase involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
T akt, by Cell Signaling Technology (3 mentions)
P gsk3β, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Phospho akt p akt, by Cell Signaling Technology (1 mentions)
Odyssey 2 colour infrared laser imaging system, by LI COR (1 mentions)
Runx2, by Boster Bio (1 mentions)
α sma, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Desmin, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Active β catenin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Image station 440cf, by Kodak (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using t gsk3β
1
Western Blot Analysis of Osteogenic Markers
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from the cells with lysis buffer containing protease inhibitors (Roche, UK). The protein concentration was measured by a BCA-200 protein assay kit (Pierce, USA). Equal amounts of proteins were separated by 4-12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5% fat-free milk for 2 h and probed with primary antibodies, p-GSK3β (1 : 1000; Cell Signaling), t-GSK3β (1 : 1000; Cell Signaling), active β-catenin (1 : 1000; Cell Signaling), myosin (1 : 500; Sigma), α-SMA (1 : 500; Sigma), desmin (1 : 500, Sigma), and GAPDH (1 : 1000; Cell Signaling) overnight at 4°C and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG diluted 1 : 2000 (Cell signaling). Protein bands were visualized on an X-ray film by using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The relative protein expression intensities were quantified by densitometry using Quantity One analysis software.
3
Western Blot Analysis of Phospho-GSK3β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (cOmplete - protease inhibitor cocktail tablets, and PhosSTOP - phosphatase inhibitor cocktail tablets; Roche), and total protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL). Cell lysates containing equal amounts of total protein (∼5–10 µg) were heated at 100°C for 5 minutes in laemmli buffer (Sigma-Aldrich), electrophoretically separated on a 10% SDS-polyacrylamide gels (Criterion Precast Gel; Bio-Rad, Hercules, CA), and transferred onto polyvinylidene difluoride (PVDF) membranes (Immun-Blot; Bio-Rad). Membranes were incubated with primary antibodies for phospho-GSK3β-Ser9 (p-GSK3β-S9; Cell Signaling Technology, Danvers, MA; 5558), GSK3β (t-GSK3β; Cell Signaling Technology; 9832) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam; ab8245). Appropriate horseradish peroxidase-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL) were used. Membranes were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and visualized using a Kodak Image Station 440CF.
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Treated cells were lysed in radioimmunoprecipitation assay buffer containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich). In order to detect cytochrome c (cyto c) release, cytoplasmic extracts were prepared as described previously by Yang et al (20 (link)). A total of 30 μg protein was separated using 10–12% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (pore size, 0.45 μm; Bio Basic, Inc., Markham, ON, Canada). The transferred membranes were then blotted with antibodies against phosphorylated (P)-ERKs, total (T)-ERKs, P-AKT, T-AKT, P-glycogen synthase kinase-3β (GSK3β), T-GSK3β, B-cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax), cyto c and GAPDH at dilutions of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 3 h at 4°C. Chemiluminescence was detected using enhanced chemiluminescence detection kits (GE Healthcare, Amersham, UK). The intensity of the bands was quantified by scanning densitometry using Quantity One 4.5.0 software (Bio-Rad Laboratories, Inc.).
5
Quantifying Tau Pathology in Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of tau were analyzed using lysates that were extracted and fractioned into soluble and insoluble fractions by ultracentrifugation utilizing the posterior half of the right hemi-brain that includes the neocortex and hippocampus [20 (link)]. Protein (20 µg/lane) from the insoluble fraction was loaded onto 4–12 % SDS/PAGE gels and blotted onto PVDF membranes, incubated mouse monoclonal antibodies against total Tau (tTau 1:1000), 3R tau (1:2000), p-tau (PHF-1 1:1500), t-GSK3β (1:500, Cell Signaling), p-GSK3β (GSK3βY216, 1:500, Life Technologies), t-Akt (1:1000, Cell Signaling), p-Akt (Ser473, 1:500, Santa Cruz) followed by HRP-tagged secondary antibodies (1:5000 Santa Cruz Biotechnology). Bands were visualized by enhanced chemiluminescence (ECL, PerkinElmer, Boston, MA) and analyzed with a quantitative Versadoc XL imaging apparatus (BioRad). β-Actin (1:3000, Sigma) was used as the loading control.
6
Western Blot Analysis of Insulin Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and blotted on polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% milk (170-6404, Bio-Rad Laboratories, Inc.) and incubated with specific primary antibodies: PTBP1 (Thermo Fisher/Invitrogen, 32-4800, 1:1000), AdipoR1 (Santa Cruz Biotechnology, sc-518030,1:500), P-IR (Cell Signaling Technology, 3024S, 1:1000), t-IR (Cell Signaling Technology, 3025S, 1:1000), P-AKT (Cell Signaling Technology, 9271S, 1:1000), t-AKT (Cell Signaling Technology, 4691S, 1:1000), P-GSK3β (Cell Signaling Technology, 9336S, 1:1000), t-GSK3β (Cell Signaling Technology, 9315S, 1:1000), GAPDH (OriGene, TA802519, 1:3000), β-ACTIN (OriGene, TA811000, 1:3000), then reprobed with appropriate horseradish peroxidase–conjugated secondary antibodies. Blots were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (SD251210, Thermo Fisher Scientific, Inc.) Phosphorylation PTBP1 antibody was constructed by ProMab Biotechnologies (Hunan China) used the antigen as a synthetic peptide GTKRGSDELF (PTB1 amino acids 11-20) with Ser-16 phosphorylated.
7
Quantification of Liver Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of protein in liver lysates was determined using Bradford methods (Bio-Rad Laboratories, Hercules, CA, USA), and aliquots of tissues was mixed with the sample buffer containing SDS and β-mercaptoethanol and heated at 95℃ for 6 min. Respective samples were then loaded to SDS-PAGE gel and subjected to electrophoresis. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare BioSciences Co., Piscataway, NJ, USA) in the transblot chamber with Tris buffer. The membrane was blocked for 1 h at room temperature with 5% skim milk in TBS-Tween 20 buffer, followed by an overnight incubation at 4℃ in diluted polyclonal antibodies of p-Akt (Ser 473) (1:2000, Cell Signaling Technology, MA, USA), t-Akt (1:2000, Cell Signaling Technology), β-actin (1:3000, Sigma-Aldrich, St Louis, MO, USA), β-tubulin (1:2000, Santa Cruz Biotechnology, CA, USA), p-GSK-3β (Ser9) (1:2000, Cell Signaling Technology), t-GSK-3β (1:2000, Cell Signaling Technology), and Nitrotyrosine (1:2000, Santa Cruz Biotechnology) Positive immunoreactive bands were quantified using the densitometer (LAS-3000, FUJIFILM Corporation, Tokyo, Japan) and normalized by β-actin or β-tubulin.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!