Phospho p53 ser15

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Phospho-p53 (Ser15) is a specific antibody that detects p53 protein phosphorylated at serine 15. This antibody can be used to analyze the phosphorylation status of p53, which is an important cellular protein involved in the regulation of various cellular processes.

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91 protocols using phospho p53 ser15

Western Blot and Flow Cytometry Assay for p53

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used in the western blotting or flow cytometry experiments: p53 (sc-6243), TFIIH p89 (sc-293), and p21 (sc-397; Santa Cruz Biotechnology); Phospho-Ser15-p53 (#9284), Phospho-Ser45-53 (#2521), and Acetyl-Lys382-p53 (#2525; Cell Signaling Technology); HRP-conjugated secondary antibodies (Bio-Rad), and Alexa Fluor-labelled secondary antibodies (Life Technologies).

Ticha I., Hojny J., Michalkova R., Kodet O., Krkavcova E., Hajkova N., Nemejcova K., Bartu M., Jaksa R., Dura M., Kanwal M., Martinikova A.S., Macurek L., Zemankova P., Kleibl Z, & Dundr P. (2019). A comprehensive evaluation of pathogenic mutations in primary cutaneous melanomas, including the identification of novel loss-of-function variants. Scientific Reports, 9, 17050.

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Quantitative Western Blot Analysis

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Western blotting was performed as previously described (Van Hee et al., 2015 (link)). Primary antibodies were: a rabbit polyclonal against GLUT1 (#652; Abcam, Cambridge, United Kingdom); rabbit monoclonals against GLUT3 (#191071; Abcam), HK 1/2 (#2024/2867; Cell Signaling, Leiden, The Netherlands), PKM2 (#4053; Cell Signaling) p21 Waf1/Cip1 (12D1) (#2947; Cell Signaling), p53 (7F5) (#2527; Cell Signaling), ATR (#2790; Cell Signaling), phospo-Ser428-ATR (#2853; Cell Signaling), ATM (D2E2) (#2873; Cell Signaling), phospho-Ser1981-ATM (D6H9) (#5883; Cell Signaling); and mouse monoclonals against phospho-Ser15-p53 (#9286; Cell Signaling) and β-actin (#A5441; Sigma-Aldrich). Secondary antibodies were HRP-coupled goat anti-rabbit and anti-mouse (Jackson ImmunoResearch, Huissen, The Netherlands). Staining was revealed with an Amersham Imager 600 (GE Healthcare, Diegem, Belgium). Data were analyzed using the ImageJ software (NIH, Bethesda, United States).

Grasso D., Medeiros H.C., Zampieri L.X., Bol V., Danhier P., van Gisbergen M.W., Bouzin C., Brusa D., Grégoire V., Smeets H., Stassen A.P., Dubois L.J., Lambin P., Dutreix M, & Sonveaux P. (2020). Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells. Frontiers in Pharmacology, 11, 263.

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Western Blot Analysis of DNA Damage Response

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Cells were washed with ice-cold PBS and incubated for 20 min on ice in 0.1% Triton X-100 lysis buffer (20 mM Tris HCl pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.1% Triton X-100; 1 mM Phenylmethanesulfonyl fluoride; 10 mM NaF; 1 mM Na3VO4, supplemented with protease inhibitor cocktail). Cells were then centrifuged at 14,000× g for 15 min at 4 °C to remove any cellular debris. Protein lysates were subsequently quantified using DC protein assay (Bio-Rad), loaded in 4–12% NuPAGE Bis-Tris Protein Gels (Thermo Fisher Scientific) according to the manufacturer’s instructions, and transferred onto Hybond ECL nitrocellulose membranes. Blocking was performed with 5% Nonfat dried milk (PanReac AppliChem, Darmstadt, Germany) for 45 min at RT. Membranes were then incubated O/N at 4°C with the following antibodies: BAP-1 (Santa Cruz Biotechnology, sc-28383); phospho(Ser345) Chk1 (Cell Signaling, Danvers, MA, USA, 2348); phospho(Thr68) Chk2 (Cell Signaling, 2197); phospho(Ser15) p53 (Cell Signaling, 9286); GAPDH (Cell Signaling, 5174); cleaved Caspase3 (Cell Signaling, 9661); phospho(Ser139)-Histone H2A.X (Cell Signaling, 9718); rabbit IgG, HRP-linked (Cell Signaling, 7074); mouse IgG, HRP-linked (Cell Signaling, 7076). Proteins were detected with horseradish peroxidase-conjugated secondary antibodies and Pierce™ ECL Western Blotting Substrate.

Anobile D.P., Bironzo P., Picca F., Lingua M.F., Morena D., Righi L., Napoli F., Papotti M.G., Pittaro A., Di Nicolantonio F., Gigliotti C., Bussolino F., Comunanza V., Guerrera F., Sandri A., Leo F., Libener R., Aviles P., Novello S., Taulli R., Scagliotti G.V, & Riganti C. (2021). Evaluation of the Preclinical Efficacy of Lurbinectedin in Malignant Pleural Mesothelioma. Cancers, 13(10), 2332.

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Comprehensive Molecular Profiling of Autophagy

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Unless otherwise mentioned reagents were from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest analytical grade. Antibodies against AMPK, phospho-Thr172-AMPK, LC3B, GAPDH, Golgin 97, Rab7, PARP, phospho-Ser780 pRb, and phospho-Ser15 p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against V-ATPase B2 subunit, E-cadherin, p21, and LAMP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibody against β-actin was from Sigma-Aldrich. Antibodies against alpha-adaptin 2, MT1-MMP (MMP14) and p62 were from Abcam (Cambridge, UK). HIF-1α and p150^Glued antibodies were from BD Biosciences (San Jose, CA, USA). Antibodies against Giantin and HSP47 were from Enzo Lifesciences (Farmingdale, NY, USA). HRP-conjugated secondary antibodies for Western blotting (goat-anti-mouse (GAM) and goat-anti-rabbit (GAR)) were from DAKO (Glostrup, Denmark). Rhodamin phalloidin, Alexa Fluor 568 conjugated GAM, and Alexa Fluor 488 conjugated GAR secondary antibodies for immunofluorescence were from Invitrogen (Carlsbad, CA, USA). Antibody against Cortactin was from Merck (Darmstadt, Germany). Concanamycin A, Forskolin, and antibody against TCIRG1 (V-ATPase a3 subunit) were from Sigma-Aldrich (St. Louis, MO, USA).

Flinck M., Hagelund S., Gorbatenko A., Severin M., Pedraz-Cuesta E., Novak I., Stock C, & Pedersen S.F. (2020). The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells. Cells, 9(2), 465.

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Antibodies Used for IP and Western Blot

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Antibodies against the following proteins were used for IPs and Western blots: CBP80 (36 (link)), eIF4E (610269, BD Bioscience; 2067, Cell Signaling Technology), UPF1 (a gift from Lynne E. Maquat), phospho-(S/T)Q (2851, Cell Signaling Technology), UPF2 (36 (link)), STAU1 (A303–956A, Bethyl Laboratories), IMPα (A300–484A, Bethyl Laboratories), PABPN1 (A303–524A, Bethyl Laboratories), eIF4A3 [sc-365549, Santa Cruz Biotechnology; (36 (link))], Y14 (MAB2484, Abnova), p53 (2524, Cell Signaling Technology), phospho-Ser15-p53 (9284, Cell Signaling Technology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), FLAG (A8592, MilliporeSigma), HA (11 867 431 001, Roche), Myc (OP10L, Calbiochem), β-actin (A5441, MilliporeSigma), and GAPDH (LF-PA0212, Ab Frontier). For IP of endogenous eIF4E, we employed an in-house anti-eIF4E antibody, which was raised in rabbits against a human eIF4E-specific peptide (MATVEPETTPTPNPPTTEEEKTESNQEVANPEHYIKH).
Signal intensities of Western blot bands were quantitated in the Image J software (version 1.5, National Institutes of Health).

Jeong K., Ryu I., Park J., Hwang H.J., Ha H., Park Y., Oh S.T, & Kim Y.K. (2019). Staufen1 and UPF1 exert opposite actions on the replacement of the nuclear cap-binding complex by eIF4E at the 5′ end of mRNAs. Nucleic Acids Research, 47(17), 9313-9328.

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Western Blot Analysis of DNA Repair Proteins in ccRCC

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Cultured cells or cells from human ccRCC and matched normal tissues were lysed with 0.5% NP-40 buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, and a mixture of protease inhibitors (Sigma-Aldrich). After centrifugation at 13,800 × g and 4 °C for 15 min, supernatants were collected for western blotting according to standard procedures. Antibodies against DNA-PKcs (#38168, 1: 1000), phospho-Ser2056 DNA-PKcs (#68716, 1: 1000), H₂AX (#7631, 1: 1000), γ-H₂AX (#9718, 1: 1000), p53 (#9282, 1: 1000), phospho-Ser15 p53 (#9284, 1: 1000), KU70 (#4103, 1: 1000), and KU80 (#2753, 1: 1000) were purchased from Cell Signaling Technology. Antibody against NNMT was purchased from Abcam (#ab58743, 1: 1000). Antibody against Actin was purchased from Genscript (#A00702, 1: 800). Anti-K-Hcy antibody (1: 1000) was generated as described previously^{56 (link)}. Chemiluminescence was measured on a Typhoon FLA 9500 instrument (GE Healthcare).

Qu Y., Feng J., Wu X., Bai L., Xu W., Zhu L., Liu Y., Xu F., Zhang X., Yang G., Lv J., Chen X., Shi G.H., Wang H.K., Cao D.L., Xiang H., Li L., Tan S., Gan H.L., Sun M.H., Qiu J., Zhang H., Zhao J.Y., Ye D, & Ding C. (2022). A proteogenomic analysis of clear cell renal cell carcinoma in a Chinese population. Nature Communications, 13, 2052.

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Western Blot Analysis of Cell Signaling

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Proteins were extracted using RIPA buffer with 1% Protease inhibitor cocktail (Nacalai Tesque). After determination of the protein concentration using Protein Quantification Assay (Takara Bio Inc.), all samples were denatured in Laemmli sample buffer for 5 min at 100 °C. The denatured samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% milk or 5% BSA, the membranes were incubated with the primary antibodies as follows: p16^INK4a (1:1000, IBL, 11104), p21^Cip1/Waf1 (1:1000, Cell Signaling Technology, 2947), Lamin B1 (1:5000, Abcam, ab16048), RB (1:500, Santa Cruz, sc-102), phospho-RB (Ser780) (1:1000, Cell Signaling Technology, 9307), p53 (1:1000, Santa Cruz, sc-6243), phospho-p53 (Ser15) (1:1000, Cell Signaling Technology, 9284) and α-Tubulin (1:2000, Sigma-Aldrich, T9026). The membranes were then incubated with the secondary antibodies as follows: anti-mouse IgG (1:2000, Cell Signaling Technology, 7076), anti-rabbit IgG (1:2000, Cell Signaling Technology, 7074) and visualized with Amersham ECL prime/select (GE Healthcare), followed by detection with chemiluminescence using LAS-3000mini imaging system (Fujifilm) and by analysis of data using Multi Guage V3.1 (Fujifilm). Uncropped and unprocessed scans of the blots are included in the Source data file.

Okumura S., Konishi Y., Narukawa M., Sugiura Y., Yoshimoto S., Arai Y., Sato S., Yoshida Y., Tsuji S., Uemura K., Wakita M., Matsudaira T., Matsumoto T., Kawamoto S., Takahashi A., Itatani Y., Miki H., Takamatsu M., Obama K., Takeuchi K., Suematsu M., Ohtani N., Fukunaga Y., Ueno M., Sakai Y., Nagayama S, & Hara E. (2021). Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion. Nature Communications, 12, 5674.

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Immunoblotting Antibodies and Reagents

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Primary antibodies for immunoblotting include Flag (Sigma, St Louis, MO, USA; M2); phospho-p53 (Ser15) (Cell Signaling, #9284; Danvers, MA, USA); and GAPDH (FL-335), β-actin (N-21), GFP (B-2), Myc (9E10), Wip1 (F-10), MDM2 (SMP14 and 2A10) and p53 (DO1 and FL393) (Santa Cruz Biotechnology). LZAP custom antiserum was previously described. Other reagents include normal IgG (Promega), HRP-conjugated secondary antibodies (Promega), goat anti-mouse IgG (H+L) secondary antibody (Dylight 550 conjugate) and goat anti-rabbit IgG (H+L) secondary antibody (DyLight 650 conjugate) (ThermoFisher), zeocin (Invitrogen, Carlsbad, CA, USA), MG132 (Sigma), carboplatin (Sigma), doxorubicin (Selleckchem, Houston, TX, USA) and paclitaxel (Sigma), nutlin (Santa Cruz Biotechnology).

Wamsley J.J., Gary C., Biktasova A., Hajek M., Bellinger G., Virk R., Issaeva N, & Yarbrough W.G. (2017). Loss of LZAP inactivates p53 and regulates sensitivity of cells to DNA damage in a p53-dependent manner. Oncogenesis, 6(4), e314-.

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DNA Damage Response Pathway Antibodies

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Antibodies to caspase-3,
caspase-8, caspase-9,
PARP, phospho-ATM (Ser1981), ATM, phospho-ATR (Ser428), phospho-Chk1
(Ser345), phospho-Chk2 (Thr68), Chk2, phospho-H2AX (Ser139), and phospho-p53
(Ser15) were purchased from Cell Signaling Technology. Antibodies
against ATM, ATR, Chk1, and PUMA were from Santa Cruz Biotechnology.
Antibodies against Topo I, Topo IIα, Topo IIβ, p53, FADD,
BAX, Bcl-xL, and Bcl-2 were from BD Biosciences. Antibody to β-actin
was purchased from EMD Millipore.

Wang M.J., Liu Y.Q., Chang L.C., Wang C.Y., Zhao Y.L., Zhao X.B., Qian K., Nan X., Yang L., Yang X.M., Hung H.Y., Yang J.S., Kuo D.H., Goto M., Morris-Natschke S.L., Pan S.L., Teng C.M., Kuo S.C., Wu T.S., Wu Y.C, & Lee K.H. (2014). Design, Synthesis, Mechanisms of Action, and Toxicity of Novel 20(S)-Sulfonylamidine Derivatives of Camptothecin as Potent Antitumor Agents. Journal of Medicinal Chemistry, 57(14), 6008-6018.

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Antibody Characterization for Cell Signaling

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Primary antibodies used for western blot experiments: phospho-p53 Ser15, cleaved Caspase-3 and cleaved PARP (Cell Signaling, Leiden, The Netherlands), MAPK15 (custom preparation), p53 (Santa Cruz Biotechnology), ACTB (Sigma Aldrich), CDKN1A/p21 (Epitomics, Burlingame, CA), LC3B (Novus Biologicals, Cambridge, UK), SQSTM1/p62 (BD Biosciences, Milan, Italy), GADD45a (Cell Signaling), phospho-ATR (Cell Signaling), phospho-CHK2 (Cell Signaling), PCNA (Cell Signaling). Primary antibodies used for immunofluorescence experiments: γH2A.X (Cell Signaling), 53BP1 (Novus Biologicals) LC3B (MBL, Woburn, MA). Primary antibody used for immunohistochemistry experiments: MAPK15 (custom preparation). Secondary antibodies used for western blot experiments: HRP-conjugated anti-Mouse IgG and HRP-conjugated anti-Rabbit IgG (Santa Cruz Biotechnology). Secondary antibodies used for immunofluorescence experiments: AlexaFluor488-conjugated anti-Rabbit IgG and AlexaFluor555-conjugated anti-Mouse IgG (Life Technologies).

Rossi M., Colecchia D., Ilardi G., Acunzo M., Nigita G., Sasdelli F., Celetti A., Strambi A., Staibano S., Croce C.M, & Chiariello M. (2016). MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors. Oncotarget, 7(15), 20981-20998.

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