Western blot analysis was performed using standard procedures for whole-cell extracts from cell lines. Antibodies used include Chk2 (1:5000, Becton Dickinson and Millipore), SIRT1 (1:2000, Millipore), acetyl-lysine (1:1000), phospho-CHK2-T68 (1:1000), phospho-CHK2-Thr387 (1:1000), phospho-p53-Ser20 (1:1000), acetyl-p53-Thr382 (1:1000), phospho-histone H2A.X (Ser139) (1:1000), phospho-ATM-Ser1981 (1:1000), ATM (1:1000), phospho-histone H3-S10 (1:1000), p-CDC25C (ser216) (1:1000), CDC25C (1:1000), cleaved PARP-1 (1:1000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology), phospho-CHK2-Thr432 (1:500, Invitrogen), P53 (Do-1, 1:1000, Santa Cruz Biotechnology), FLAG (clone M2) (1:2000), α-tubulin (1:5000, Sigma), and β-actin (1:5000, Sigma). For immunoprecipitation analysis, cell lysates (1–4 mg) after preclearing were mixed with antibodies (2 μg) at 4 °C overnight followed by the addition of 30 μl of protein-G (for mouse antibodies)- or protein-A (for rabbit antibodies)-coupled sepharose beads (GE) for 3 h at 4 °C. Immune complexes were washed three times with lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, protease, phosphatase inhibitor mixture (Sigma)]. After boiling in 2× loading buffer, samples were subjected to SDS-PAGE, and then scanned using ECL.
Phospho p53 ser20
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-p53-Ser20 is a lab equipment product that detects the phosphorylation of p53 at serine 20. It is used to measure the activation and regulation of the p53 protein, which plays a crucial role in cellular processes such as DNA repair, cell cycle control, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Phospho atm ser1981, by Cell Signaling Technology (2 mentions)
Cdc25c, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Phospho histone h2a x ser139, by Cell Signaling Technology (1 mentions)
Phospho histone h3 s10, by Cell Signaling Technology (1 mentions)
Sirt1, by Merck Group (1 mentions)
Acetyl lysine, by Merck Group (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Flag clone m2, by Merck Group (1 mentions)
Protease phosphatase inhibitor mixture, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
P cdc25c ser 216, by Cell Signaling Technology (1 mentions)
P53 do 1, by Santa Cruz Biotechnology (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Phospho chk1 ser345, by Cell Signaling Technology (1 mentions)
Phospho cdc25c ser216, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
5 protocols using phospho p53 ser20
1
Western Blot Analysis of Cell Signaling
2
Cucurbitacin B Molecular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cucurbitacin B, purchased from ShunBo Biological Engineering Technology Co., Ltd (Shanghai China), was dissolved in dimethyl sulfoxide (DMSO) to make 200 µM stock solutions and was kept at −20°C. The stock solutions were freshly diluted to the desired concentration just before use. N-acetyl-L-cysteine (NAC) was purchased from Beyotime (Haimen, China). Specific antibodies against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phospho-p53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA). Phospho-Cdk1-Tyr-15, Cyclin B1, 14-3-3-σ were from Santa Cruz Biotechnology (USA). Cdk1 antibody was obtained from BD Transduction Laboratories™ (USA). Antibodies for ATM and γH2AX were obtained from GeneTex and Millipore respectively.
3
Signaling Pathway Modulation in PEDV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
p38 inhibitor SB203580 (10 μM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μM), extracellular signal-regulated kinase (ERK) inhibitor PD98059 (5 μM), PKA inhibitor H-89 (5 μM), c-Jun inhibitor SR11302 (5 μM), CREB inhibitor EML-425 (5 μM), and p53 inhibitor PFT-α (10 μM) are all purchased from MedChemExpress (Shanghai, China). All inhibitors were configured with dimethyl sulfoxide (DMSO; Solarbio, Beijing, China). Antibodies against Bax, Bcl-2, caspase 3, caspase 8, caspase 9, FasL, Fas, p53, his, phospho-p53(ser15), phospho-p53(ser46), and phospho-p53(ser20) were purchased from Cell Signaling Technology (Danvers, MA, United States). Monoclonal antibodies against CCN1, c-Jun, phospho-c-Jun, c-Fos, phospho-c-Fos, CREB, and phospho-CREB were obtained from ABclonal (Wuhan, China). Antibodies against PEDV (CH/SXYL/2016) N protein were stored in our laboratory (Xu et al., 2019 (link)).
4
Quantitative Protein Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses were performed as previously described (22 (link)). Immunoblotting was carried out with antibodies against human BRCA-1 (catalog no. 9010), DNMT-1 (catalog no. 5119), cyclin D1 (catalog no. 92G2), phospho-p53-(Ser20) (catalog no. 9287), and GAPDH (catalog no. 2118) obtained from Cell Signaling Technology, and ERα (catalog no. sc-542) obtained from Santa Cruz Biotechnology. Immunocomplexes were detected by using enhanced chemiluminescence (GE Healthcare Life Sciences). The GAPDH protein was used as an internal control for normalization of protein expression.
5
Cyproheptadine's Effects on Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 and Huh-7 were seeded in 6-well plates at 2 × 105 cells per well and cultured for 24 h, starved in medium without FBS for 24 h, and then treated with 40 μM cyproheptadine for various durations. Total cellular proteins were extracted, and protein concentration was determined for the extracts using the Bio-Rad Protein Assay reagent (Bio-Rad) with bovine serum albumin as a standard. Each lysate (10 μg) was resolved on denaturing polyacrylamide gels and transferred electrophoretically to PVDF transfer membranes. After blocking with 3% blocker (Bio-Rad) in Tris-buffered saline with Tween 20 (TBST), the membranes were incubated at room temperature for 2 h with primary antibodies—1:5000 diluted antibody against GAPDH; 1:1000 diluted antibody against PARP, p21, p27, Rb (D20), phospho-Rb (Ser795), cyclin D1, p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), CHK2, phospho-CHK2 (Thr68), p53 (7F5), or phospho-p53 (Ser20) (Cell Signaling, Danvers, MA); or 1:1000 diluted antibody against p16INK4A or HBP1 (Millipore, Temecula, CA). Immunoreactive proteins were detected by incubation with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. After washing with TBST, the reactive bands were developed with an enhanced chemiluminescent HRP substrate detection kit (Millipore, Billerica, MA) and identified using the BioSpectrum 800 imaging system (UVP).
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!