After washing, cold RIPA with protease inhibitors (Roche) was used to lyse cells for 15 min. Then, after centrifugation, the cell supernatant was kept frozen at −80°C until use. Bradford assays (Bio-Rad Laboratories; CA, USA) were employed to assess protein concentrations. Samples (20 μg) were separated via SDS-PAGE, after which dry transfer to nitrocellulose membranes was conducted. After being blocked in 5% BSA for 1 h, primary antibodies were used to probe the membranes overnight at 4°C, including antibodies targeting human SIRT1, acetylated-p53 at K382 (Ac-p53), p21, forkhead box O1 (FoxO-1) (Cell Signaling Technology; USA), Ac-FoxO-1, total p53 (1: 2000 dilution), and senescence marker protein-30 (SMP-30) (all from Santa Cruz Biotechnology). β-actin served as an internal control and was purchased from Sigma Aldrich. After incubation with the appropriate secondary antibodies (1: 10 000 dilution; GE Healthcare, Buckinghamshire, UK), the immunostained protein bands were visualized with an ECL system (ProteinSimple; Santa Clara, USA), followed by quantification using ProteinSimple image software. All samples had 3 biological replicates, and each biological replicate was assayed in duplicate; therefore, the data for each time point are an average of 6 individual replicate runs. Representative images of the immunostained bands are presented in the figures.
Smp30
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The SMP30 is a laboratory equipment designed for sample preparation and processing. It is a multi-purpose instrument that can be used for a variety of applications. The core function of the SMP30 is to provide consistent and reliable sample preparation, ensuring the integrity and accuracy of subsequent analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
α sma, by Thermo Fisher Scientific (3 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Cyclin e, by Santa Cruz Biotechnology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Ac foxo1, by Santa Cruz Biotechnology (1 mentions)
Ac p53, by Cell Signaling Technology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Phospho akt, by Santa Cruz Biotechnology (1 mentions)
Twist, by Santa Cruz Biotechnology (1 mentions)
C caspase3, by Santa Cruz Biotechnology (1 mentions)
Phospho fak, by Santa Cruz Biotechnology (1 mentions)
7 protocols using smp30
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The whole lysates (30-50) μg of sample protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8-12 % gel, and transferred to polyvinylidene fluoride membranes. After blocking with 5 % skim milk in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05 % Tween-20), the membranes were immunoblotted with the primary antibodies against CK2α (1:1000), Pfn1 (1:1000), Myosin-X (1:1000), Myosin IIA (1:1000) (NOVUS, Littleton, CT, USA), fibronectin (1:1000), α-SMA (1:1000) (Thermo Fisher Scientific), p-Myosin IIA (1:1000), p-P53 (1:1000), P16 (1:1000) (Cell signaling, Danvers, MA, USA), P21 (1:1000), SMP30 (1:1000), cyclin D1 (1:1000), CDK4 (1:1000), cyclin E (1:1000), CDK2 (1:1000), and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed, and the primary antibodies were detected by means of goat anti-rabbit IgG (1:10,000) or goat anti-mouse IgG antibodies (1:10,000) (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Thermo Fisher Scientific). Band densitometric quantifications were determined using ImageJ software 1.48v.
3
Western Blot Analysis of Stem Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from MSCs using RIPA lysis buffer (Thermo Fisher Scientific). Cell lysates and tissue homogenates (30 μg protein) in sample buffer were separated by electrophoresis on an 8–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane for probing with antibodies. After washing with Tris-buffered saline/Tween-20 buffer (0.05% Tween-20, 150 mM NaCl, 10 mM Tris-HCl; pH 7.6), membranes were blocked with 5% bovine serum albumin for 1 h at room temperature then incubated with primary antibodies against SMP30, p21, CDK2, CDK4, Cyclin E, Cyclin D1, phospho-FAK, phospho-Akt, TWIST, PrPC, c-Caspase3, β-actin, and GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were detected using enhanced chemiluminescence reagents (GE healthcare).
4
Protein Expression Analysis of MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs homogenates were extracted by using RIPA lysis buffer (Thermo Scientific). Cell lysates (20 μg of total protein) were separated by using 6–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membrane (Thermo Scientific). After the blots had been washed with a solution comprising 10 mM Tris-HCl (pH 7.6; Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), and 0.05% Tween-20 (Sigma-Aldrich), the membranes were incubated with 5% bovine serum albumin for 1 h at room temperature and then, incubated with appropriate primary antibodies against phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), phosphorylated 5′-AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3, beclin 1, ATG7, p62/Sequestosome 1 (p62/SQSTM1), senescence marker protein 30 (SMP30), and β-actin (all from Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed more than twice, and the primary antibodies were detected using a goat anti-rabbit IgG or a goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
5
Quercetin Modulates AMPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quercetin and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p-LKB1, AMPK, p-AMPK, p-acetyl CoA carboxylase (ACC), p53, Bax, cytochrome C, caspase-3, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p21, p16, Bcl-2, SMP30 and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-Bcl-2 was purchased from GeneTex (Irvine, CA, USA). Compound C, an AMPK inhibitor, was provided by Calbiochem (La Jolla, CA, USA). Control and AMPK siRNA were purchased from Santa Cruz Biotechnology.
6
Antcins Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antcin A, antcin B, antcin C, antcin H, antcin K and antcin M were isolated from the fruiting bodies of A. cinnamomea and A. salmonea as described previously [17 (link)]. The purity of the antcins was above 99% as confirmed by HPLC and FT-NMR analysis. Minimum essential medium (MEM), Medium 199 (M-199), fetal bovine serum (FBS), sodium pyruvate, penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA). Heparin sodium salt, endothelial cell growth supplement (ECGS), N-acetylcysteine, 2′, 7′-dichlorofluorescein diacetate (DCFH2-DA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and D-Glucose were purchased from Sigma-Aldrich (St Louis, CA). Antibodies against cyclin D1, cyclin B1, cyclin E, CDK2, CDK4, CDK6, Cdc2, phos-pRb, p16INK4A, p21CIP1, acety-p52, phos-p53, phos-FoxO1, FoxO1, phos-JNK/SAPK, JNK/SAPK, phos-p38 MAPK, p38 MAPK, phos-ERK1/2, ERK1/2, Phos-AKT, AKT, histone H3, phos-SIRT-1, SIRT-1, SIRT-3, SIRT-6 and Keap-1 were obtained from Cell Signaling Technology, Danvers, MA. Antibodies against p53, SMP30 and acetyl-FoxO1 were purchased from Santa Cruz Biotechnology, Dallas, TX. Antibodies against HO-1 and NQO-1 were obtained from Abcam, Cambridge, UK. All other chemicals were reagent grade or HPLC grade and supplied by either Merck (Darmstadt, Germany) or Sigma-Aldrich.
7
Senescence Regulation by PPARδ Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chloramethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCF-DA, Cat#287810), resveratrol (Cat#554325), and WY-14643 (Cat#681725) were purchased from Calbiochem (La Jolla, CA, USA). GW501516 (Cat#ALX-420-032) was purchased from Enzo Life Sciences (Farmingdale, NY, USA) Rosiglitazone (5-[[4-(2-[methyl-2-pyridinylamino]ethoxy)phenyl]methyl]-2,4-thiazolidinedione, Cat#71740) was provided by Cayman Chemical Company (Ann Arbor, MI, USA). An anti-β-actin (Cat#A2066) antibody, glucose (Cat#G8644), a senescence-associated β-galactosidase (SA β-gal, Cat#CS0030) staining kit, sirtinol (Cat#S7942), and lentiviral particles expressing non-targeting control (pLKO.1-puro Non-Target shRNA Control Transduction Particles, Cat#SHC001V) and PPARδ-targeting (TRCN0000350974, MISSION Lentiviral Transduction Particles) small hairpin RNA (shRNA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A monoclonal antibody specific for phosphorylated H2A histone family member X (γ-H2A.X, Cat#2577) and goat anti-rabbit IgG conjugated to Cy3 (indocarbocyanine, Cat#A10520) were purchased from Cell Signaling Technology (Danvers, MA, USA) and Invitrogen (Waltham, MA, USA), respectively. Monoclonal antibodies specific for p21 (Cat#SC-6246), p53 (Cat#SC-126), and SMP-30 (Cat#SC-130344), and a polyclonal antibody specific for SIRT1 (Cat#SC-19857) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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!