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Phospho p53

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Phospho-p53 is a primary antibody that specifically recognizes p53 protein phosphorylated at specific serine residues. It is used to detect the phosphorylation status of p53, a key regulator of cellular responses to various stress stimuli.

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Lab products found in correlation

Phospho chk2, by Cell Signaling Technology (10 mentions) Gapdh, by Cell Signaling Technology (10 mentions) Phospho chk1, by Cell Signaling Technology (7 mentions) Ripa buffer, by Thermo Fisher Scientific (7 mentions) β actin, by Merck Group (6 mentions) β actin, by Cell Signaling Technology (5 mentions) Cleaved parp, by Cell Signaling Technology (5 mentions) Caspase 3, by Cell Signaling Technology (4 mentions) Caspase 9, by Cell Signaling Technology (4 mentions) Cyclin d1, by Cell Signaling Technology (4 mentions) Phospho jnk, by Cell Signaling Technology (4 mentions) Phospho p38, by Cell Signaling Technology (4 mentions) Phospho atm, by Cell Signaling Technology (4 mentions) Phospho akt, by Cell Signaling Technology (4 mentions) Cleaved caspase 3, by Cell Signaling Technology (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions) Bcl 2, by Cell Signaling Technology (4 mentions) Actin, by Merck Group (3 mentions) Phospho mtor, by Cell Signaling Technology (3 mentions)

57 protocols using phospho p53

1

Western Blot Analysis of Signaling Proteins

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Western blotting was carried out as described.(14) For immunodetection, antibodies against the following proteins were used: Met (Cell Signaling Technology, #3127) at 1:1,000; phosphorylated (phospho)‐Met (Tyr1234/1235) (Cell Signaling Technology, #3077) at 1:1,000; GRB2 associated binding protein 1 (Gab1; Cell Signaling Technology) at 1:1,000; phospho‐Gab1 (Cell Signaling Technology) at 1:1,000; Akt (Cell Signaling Technology, #4691) at 1:1,000; phospho‐Akt (Thr308) (Cell Signaling Technology, #4060) at 1:2,000; extracellular kinase 1 and 2 (Erk 1/2; Cell Signaling Technology, #4695) at 1:1,000; phospho‐Erk 1/2 (Thr202/Tyr204) (Cell Signaling Technology, #4370) at 1:2,000; p53 (Leica, Nussloch, Germany, #NCL‐L‐p53‐CM5p) at 1:2,000; phospho‐p53 (primary target, Serine 15 [Ser15]) (Cell Signaling Technology, #9284) at 1:1,000; HGF (Abcam, #ab83760) at 1:1,000; p21 (Abcam, #ab109199) at 1:1,000; and β‐actin (Sigma‐Aldrich, A5316) at 1:10,000.
Urabe M., Hikita H., Saito Y., Kudo S., Fukumoto K., Mizutani N., Myojin Y., Doi A., Sato K., Sakane S., Makino Y., Kodama T., Sakamori R., Tatsumi T, & Takehara T. (2021). Activation of p53 After Irradiation Impairs the Regenerative Capacity of the Mouse Liver. Hepatology Communications, 6(2), 411-422.
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2

Protein Expression Analysis in Cells

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After plasma treatment for 48h, cell protein was extracted using RIPA lysis buffer (Sigma), and the concentration was determined by BCA protein assay kit (Sigma). Protein samples were denatured with 5×protein loading buffer at 95°C for 5min and separated in 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). Subsequently, proteins were transferred to 0.2 μm of polyvinylidene difluoride membranes (Millpore, Billerica, MA, USA) at 70 V for 2.5 h. After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibody and then washed with TBST three times. The horseradish peroxidase (HRP)–conjugated secondary antibodies, goat anti-rabbit IgG and anti-mouse IgG (1:2000) (Abgent, San Diego, CA, USA), were added and incubated for 1h on a horizontal orbital shaker. Images were visualized with ECL chemiluminescent substrate (Millipore, Bedford, MA, USA) using a ChemiDoc-It 510 system (UVP, Upland, CA, USA) and protein bands were analyzed by densitometric analysis using ImageJ software. Primary antibody against human included caspase-3 (1:500), caspase-8 (1:500), caspase-9 (1:500), phospho-p53 (1:200) (Cell Signaling Technology, Danvers, MA, USA), CD95 (1:200) (Santa Cruz, CA, USA) and β-actin (1:1000) (Immuno Way, Newark, DE, USA).
Xu D., Xu Y., Cui Q., Liu D., Liu Z., Wang X., Yang Y., Feng M., Liang R., Chen H., Ye K, & Kong M.G. (2018). Cold atmospheric plasma as a potential tool for multiple myeloma treatment. Oncotarget, 9(26), 18002-18017.
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3

Protein Expression Analysis Protocol

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Cells were harvested, rinsed with ice-cold phosphate buffered saline, and lysed in homogenization buffer (50 mM Tris-Cl, pH 6.8) containing protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA), 10% sodium dodecyl sulfate (SDS), and 10% glycerol. Protein concentrations of whole-cell lysates were determined using bicinchoninic acid (BCA) protein assay (Thermo Scientific, Waltham, MA, USA). Blots were probed with primary antibodies against phospho-p53 (Ser15), phospho-ETK (Tyr40), ELK1 and ETK (Cell Signaling Technology, Beverly, MA, USA); γ-H2AX, phospho-Nibrin/Nbs1 (Ser343) and Nibrin (Upstate Biotechnology, Lake Placid, NY, USA); p21, p53 and β-actin (Santa Cruz Biotechnology, California, USA); phospho-Gab2 (Tyr643), Gab2, phospho-BTK (Tyr550) and BTK (Abcam, Cambridge, MA, USA); phospho-CamK4 (Thr196/200) and CamK4 (Aviva Systems Biology, San Diego, USA). Western blotting was performed using standard protocols, and membranes were visualized by enhanced chemiluminescence (ECL solution, Amersham Biosciences, Uppsala, Sweden).
Yim J.H., Yun J.M., Kim J.Y., Lee I.K., Nam S.Y, & Kim C.S. (2017). Phosphoprotein profiles of candidate markers for early cellular responses to low-dose γ-radiation in normal human fibroblast cells. Journal of Radiation Research, 58(3), 329-340.
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4

Molecular Mechanisms of Cytotoxicity

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Convallatoxin was obtained from Pfaltz & bauer (Waterbury, CT, USA). Peruvoside was purchased from Research Plus, Inc. (Barnegat, NJ, USA). Strophanthidin, Thiazolyl blue tetrazolium bromide (MTT), Cisplatin, Sodium dichromate (Na2Cr2O7.2H2O) [Cr(VI)], Triton X-100, DNAse free RNAse and Propidium Iodide were obtained from Sigma Aldrich (St Louis, MO, USA). Antibodies for p53, phospho-p53, CDK4, Cyclin D1, PARP, XIAP, p-62, phospho-Akt (Ser 473), total-Akt, phospho-ERK, total-ERK, pEGFR, EGFR, β-Catenin, Vimentin, Slug and peroxidase-labeled secondary rabbit and mouse antibodies were obtained from Cell Signaling Technology (Denvers, MA, USA). Antibody for Bcl2 was procured from Santa Cruz Biotechnology (Dallas, TX, USA). Bicinchoninic acid and Supersignal West Pico chemiluminescent substrate was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Kaushik V., Azad N., Yakisich J.S, & Iyer A.K. (2017). Antitumor effects of naturally occurring cardiac glycosides convallatoxin and peruvoside on human ER+ and triple-negative breast cancers. Cell Death Discovery, 3, 17009-.
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5

Trx2-Mediated Oxidative Stress Response

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Ginkgolic Acid [(GA,15:1) - CAS 22910-60-7, 345887] was purchased from Millipore. N-ethylmaleimide (NEM, SENPs inhibitor, E1271), streptonigrin (SN, SENPs inhibitor, S1014) were bought from Sigma. Protein A/G PLUS-Agarose (sc-2003) was obtained from Santa Cruz.
Antibodies for immunoblotting, immunoprecipitation, and immunostaining were: V5 (Cell Signaling Technology, 13202, Rabbit, WB 1:1000), Trx2 (Abcam, ab185544, Rabbit, WB, 1:10000), Trx2 (Santa Cruz, sc-133201, Mouse, IF, 1:50), Phospho-p53 (Cell Signaling Technology, 9284, Rabbit, WB 1:500), P21 (Cell Signaling Technology, 2947, Rabbit, WB 1:1000), Phospho-Histone H2A.X (Cell Signaling Technology, 9718, Rabbit, WB 1:200), β-Actin (Cell Signaling Technology, 4970, Rabbit, WB 1:1000), α/β-Tubulin (Cell Signaling Technology, 2148, Rabbit, WB 1:1000), TFAM (Cell Signaling Technology, 8076, Rabbit, IF 1:100), PMPCA (Santa Cruz, sc-390471, mouse, IP 1:50 WB 1:500), SUMO1 (Cell Signaling Technology, 4930, Rabbit, WB 1:200), SUMO2/3 (life technologies, 519100, Rabbit, 1:600), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202, IF 1:200), Donkey anti-Rabbit IgG (H + L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A-21207, IF 1:200), normal mouse IgG (Santa Cruz, sc-2025, IP 1:100).
Chen C., Wang K., Zhang H., Zhou H.J., Chen Y, & Min W. (2019). A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity. Frontiers in Physiology, 10, 1089.
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6

Western Blot Analysis of DNA Damage Signaling

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Cells were collected, centrifuged, and re-suspended in radioimmunoprecipitation assay (RIPA) buffer plus protease inhibitors. The cells were then incubated in RIPA for 15 min at 4°C and centrifuged at 4°C for 15 min. Protein concentrations were determined using a Bradford protein assay. The samples were then resuspended in 4× lithium dodecyl sulfate sample buffer and denatured. Protein samples were electrophoresed on 4–20% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and then transferred to nitrocellulose membranes in transfer buffer (20% methanol, 11% glycine, and 3.4% Tris) at 100 V. The membranes were blocked with 5% milk in Tris-buffered saline—0.1% Tween (TBST) at room temperature for 45 min. The membranes were then incubated overnight in 5% milk in TBST at 4°C with primary antibodies. Membranes were subsequently washed with TBST and exposed to horse radish peroxidase-conjugated secondary antibodies in 5% milk in TBST at room temperature for 1 hr. The membranes were developed and enhanced using an enhanced chemiluminescent kit.
The primary antibodies used were: CtIP (14-1; (31 (link))), beta-actin (NB600-501SS, Novus), tubulin (3708-100, BioVision), Exo1 (ab95068, Abcam), DNA2 (PA5-23691, Invitrogen), phospho-Chk2 (2197S, Cell Signaling), phospho-p53 (9284, Cell Signaling) and GAPDH (sc-47724, Santa Cruz).
Stroik S., Kurtz K, & Hendrickson E.A. (2019). CtIP is essential for telomere replication. Nucleic Acids Research, 47(17), 8927-8940.
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7

Quantitative Analysis of DNA Damage Response

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Fibroblasts were transfected with 200 nM of ASOs as described above. Forty-eight hours after transfection, cells were irradiated with 1.5 Gy using a caesium-137 source, and then incubated for 60 min at 37 °C. Cells were washed in PBS, fixed in 4% (w/v) paraformaldehyde and permeabilized with 0.1% (w/v) Triton X-100 in PBS at room temperature. Cells were then incubated overnight in PBS with 3% BSA and antibodies to phospho-P53 (Cell Signaling Tech) and phospho-KAP1 (Bethyl Lab) and were visualized with immunoglobulin G Alexa Fluor conjugates (Life Technologies). DNA was counterstained with Hoechst 33342. Images were collected with the ImageXpress Micro microscope (Molecular Devices) and processed with MetaXpress (Molecular Devices). The abundance of targets expressed in nuclei was quantified.
Kim J., Woo S., de Gusmao C.M., Zhao B., Chin D.H., DiDonato R.L., Nguyen M.A., Nakayama T., Hu C.A., Soucy A., Kuniholm A., Thornton J.K., Riccardi O., Friedman D.A., El Achkar C.M., Dash Z., Cornelissen L., Donado C., Faour K.N., Bush L.W., Suslovitch V., Lentucci C., Park P.J., Lee E.A., Patterson A., Philippakis A.A., Margus B., Berde C.B, & Yu T.W. (2023). A framework for individualized splice-switching oligonucleotide therapy. Nature, 619(7971), 828-836.
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8

Molecular Mechanisms of 3-BDB and PM2.5 Exposure

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Cells were treated with 30 μΜ of 3-BDB for 1 h and then with 50 μg/mL PM2.5 for 24 h, and mice skin tissues were treated with 3-BDB and PM2.5 according to the above animal experiment method. Protein lysis buffers from the cells and mouse skin were loaded into a separating gel containing SDS-PAGE electrophoresis buffer. The target proteins were transferred onto membranes and shaken with primary and secondary antibodies sequentially. Finally, protein bands were obtained using the Amersham enhanced chemiluminescence, plus a Western blotting detection system (GE Healthcare, Buckinghamshire, UK). The primary antibodies used were as follows: actin (Sigma-Aldrich Co., Ltd.), c-Jun N-terminal kinase (JNK), p38 (Genetex Inc., Irvine, CA, USA), phospho-H2A.X, phospho-p53, caspase-9, caspase-3, mitogen-activated protein kinase kinases (MEK)1/2, phospho-MEK, phospho-extracellular regulated kinase (ERK), stress-activated ERK kinase (SEK)1, phospho-SEK, phospho-JNK, phospho-p38, c-Fos, c-Jun, phospho-c-Jun (Cell Signaling Technology, Danvers, MA, USA), B-cell lymphoma protein (Bcl)-2, Bcl-2 associated X (Bax), ERK2 (Santa Cruz Biotechnology, Dallas, TX, USA), IL-1β, matrix metalloproteinase (MMP)-2, MMP-9 (Abcam, Cambridge, MA, USA), p53, IL-6 (Invitrogen), MMP-1 (Cusabio, Houston, TX, USA).
Zhen A.X., Piao M.J., Kang K.A., Fernando P.D., Herath H.M., Cho S.J, & Hyun J.W. (2023). 3-Bromo-4,5-dihydroxybenzaldehyde Protects Keratinocytes from Particulate Matter 2.5-Induced Damages. Antioxidants, 12(6), 1307.
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9

Western Blot Analysis of Cell Signaling

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Following the experimental treatment, Western blot analysis were performed as previously described (6 (link)). Immunoblot analyses were carried out on lysates extracted from cells or tumors. Protein concentration was determined, and equal amounts of total proteins were separated on SDS-PAGE. Antibodies used included total (SCT #2602) and phospho-Pak1(SC #2606), Mek (SCT#9121), phospho-Mek pSer298(SCT#9128), Erk (SCT#9102), phospho-Erk1/2 (pThr202/pTyr204) (SCT#9101), phospho-ATM, phospho-ATR, phospho-Chk1, phospho-Chk2, phospho-p53, p21Cip SCT DNA damage sampler kit, #9947), cyclin B(SCT#4128), cyclin D1(SCT#2978), GAPDH (SCT#4138) were from Cell Signaling Technology.
Prudnikova T.Y., Villamar-Cruz O., Rawat S.J., Cai K.Q, & Chernoff J. (2015). Effects of p21-activated kinase 1 inhibition on 11q13 amplified ovarian cancer cells. Oncogene, 35(17), 2178-2185.
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10

Western Blot and Immunoprecipitation Analyses

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Western blot (WB) and immunoprecipitation (IP) analyses were performed as described previously [22 (link)]. The following antibodies were used in this study: Primary antibodies included phospho-AKT at S473 (#4060), phospho-AKT at T308 (#13038), phospho-Foxo3α at Thr32 9 (#9464), phospho-AMPKα1 at Thr172 (#2535), phospho-p70S6K at Thr389 (#9206), phospho-SIN1 at Thr86 (#14716S), phospho-NDRG1 at Thr346 (#5482), phospho-p53 at Ser6 (#9285), Ser9 (#9288), Ser15 (#9284), Ser20 (#9287), and Ser37 (#9289), phospho-pRb at Ser807/811 (#9308), mTOR (#2972), Raptor (#2280), Rictor (#2140), TSC2 (#6935), 4EBP1 (#9452), and phospho-4EBP1 at Thr37/46 (#2855) (Cell Signaling Technology, Danvers, MA, USA); PTEN (#SC-7974), MDM2 (#SC-965), phospho-PKCα at Ser657 (#SC-12356), and p21 (#SC-397) (SantaCruz, Dallas, TX, USA); p53 (#NCL-L-p53-DO7) (DO7, Leica, Milton Keynes, UK); Actin (#G043) (Applied Biological Materials Inc.); Flag (#A8592), and Catalase (#C-0979) (Sigma); Myc (#OP10) (Calbiochem, CA, USA).
Jung S.H., Hwang H.J., Kang D., Park H.A., Lee H.C., Jeong D., Lee K., Park H.J., Ko Y.G, & Lee J.S. (2018). mTOR kinase leads to PTEN-loss-induced cellular senescence by phosphorylating p53. Oncogene, 38(10), 1639-1650.
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