Ph2ax s139

Manufactured by Cell Signaling Technology

PH2AX (S139) is an antibody that recognizes phosphorylation of the serine 139 residue of the histone variant H2AX. This phosphorylation event is a marker of DNA double-strand breaks and is involved in the cellular DNA damage response.

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7 protocols using ph2ax s139

Western Blot Analysis of Cell Signaling

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Anti-pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), pChk2, pChk2 (T68), pCdc25c (S216), 53BP1, Cdc2, pCdc2 (Y15), Cyclin B1, D1 and E, PARP, pERK1/2, ERK 1/2, AKT, pAKT (S473), Bcl-X_L, GAPDH and pH2AX (S139) from Cell Signaling Technologies; pChk1 (S296), FANCF and FANCD2 from Abcam, and Bcl-2 and Mcl-1 from Santa Cruz. Treated and untreated cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.

Bryant C., Rawlinson R, & Massey A.J. (2014). Chk1 Inhibition as a novel therapeutic strategy for treating triple-negative breast and ovarian cancers. BMC Cancer, 14, 570.

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Cell Cycle Regulation Protein Analysis

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Anti- pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), Cdc2, pCdc2 (Y15), Cyclin B1 and pH2AX (S139) from Cell Signaling Technologies and pChk1 (S296) from Abcam. Treated and untreated cells were washed once with PBS and lysed in 50 mM Tris-pH6.8, 2% SDS, protease and phosphatase inhibitor cocktails (Roche) and boiled for 5 minutes. Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.

Bryant C., Scriven K, & Massey A.J. (2014). Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells. Molecular Cancer, 13, 147.

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Quantifying Cyclin E Levels in Stressed Cells

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Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin (10 μg/mL, 10 min) were fixed with 4% paraformaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: RAD51 (ab1837, Abcam, 1:200), cyclin E (4132S, Cell Signaling), pH2AX S139 (9718S, Cell Signaling), and cyclin A (ab39, Abcam). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Fisher Scientific), Alexa Fluor 488 (green) goat anti-mouse (A11001, Thermo Fisher Scientific). After washing, the cells were stained with 10 µM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described (Murthy et al., 2018 (link)). cyclin E intensity was measured for each cell. Average cyclin E intensity of cells grown in media without growth factor for 4 hr was used to define the threshold of cyclin E positive.

Hu C., Bugbee T., Palinski R., Akinyemi I.A., McIntosh M.T., MacCarthy T., Bhaduri-McIntosh S, & Wallace N. (2023). Beta human papillomavirus 8E6 promotes alternative end joining. eLife, 12, e81923.

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Quantifying mRNA and Protein Expression

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The expression of mRNA was quantified by real-time PCR using the TaqMan probes in an Applied Biosystems 7300 Real-Time PCR system (Life Technologies, Carlsbad, CA). Data were normalized to Rpl19 RNA. Western blot analysis was performed as described previously (Cho et al. 2017 (link)). Rabbit monoclonal antibodies used from Cell Signaling Technology (Danvers, MA) were Acetylated-Lysine, GCN5, and p-H2AX-S139. Mouse monoclonal antibodies used from Abcam (Cambridge, MA) were Total OXPHOS Rodent WB Antibody Cocktail and PINK1 and from Santa Cruz Biotechnology (Dallas, TX) were β-actin and TFAM. Rabbit polyclonal antibodies used were ACLY (Cell Signaling Technology), MTATP6 (Abcam), SIRT1 (Millipore, Billerica, MA), and PGC-1α (Novus Biologicals, Littleton, CO). The monoclonal antibody against human G6Pase-α has been described (Cho et al. 2017 (link)).

Cho J.H., Kim G.Y., Mansfield B.C, & Chou J.Y. (2018). Sirtuin signaling controls mitochondrial function in glycogen storage disease type Ia. Journal of inherited metabolic disease.

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Comprehensive Protein Profiling for Cancer Research

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Antibodies against IKKβ, catalase, SOD1, p21, p27, p-Chk1(S345), Chk1, p-Chk2(T68), Chk2, p-H2AX(S139), H2AX, p-ATM(S1981), ATM, MGMT, PARP, Caspase-3, p-p53(S15), p53, survivin, XIAP, cIAP-1, cIAP-2, Mre11, Rad50, p95/NBS1, were purchased from Cell Signaling (Beverly, MA). Antibodies against Bak, Bcl-2, Bcl-xL, GAPDH, and actinin were purchased from Santa Cruz Biotechnology, Inc. Fotemustine and antibody against Flag (M2) was purchased from Sigma. 8-OHdG ELISA kit, mirin and SC-514 for in vivo experiment were purchased from Abcam. SC-514 for in vitro experiment, carmustine and lomustine were purchased from Enzo Life Sciences. PLX4032, BMS-345541, TPCA-1, LY-2409881, AZD3264, IKK-16 and PS-1145 were purchased form Selleckchem ML 120B dihydrochloride and PF184 were purchased from Tocris Bioscience. Crystal violet, propidium iodide (PI), N-acetyl-L-cysteine (NAC), temozolomide, 3-Amino-1,2,4-triazole (ATZ), hydrogen peroxide solution and 3-(4,5-Dimethyl-2-thiazolyl)−2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma. 6-Carboxy-2′,7′-dichlorofluorescein diacetate (DCF) was purchased from Thermo Scientific.

Tse A.K., Chen Y.J., Fu X.Q., Su T., Li T., Guo H., Zhu P.L., Kwan H.Y., Cheng B.C., Cao H.H., Lee S.K., Fong W.F, & Yu Z.L. (2017). Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition. Redox Biology, 11, 562-576.

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Comprehensive Protein Analysis by Western Blot

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Western blot analyses were performed as per the manufacturer’s instructions using the following antibodies: ATM (A1106; Sigma-Aldrich), p-ATM S1981 (ab91292; Abcam), cleaved caspase 3 (ab32042; Abcam), mTOR(#2972 and #2983; Cell Signaling), p-mTOR S2448 (#2971; Cell Signaling), c-MYC (#5605; Cell Signaling), p-H2AX S139 (#9718; Cell Signaling), p-4E-BP1 T37/47 (#2855; Cell Signaling), p-4E-BP1 S65 (#9456; Cell Signaling), p-S6 S240/244 (#2215; Cell Signaling), H3(#9715; Cell Signaling). p-AKT S473 (#4060; Cell Signaling), and AKT (#9272; Cell Signaling). Each blot was quantified via densitometry by using Image Lab Software (Bio-Rad). Quantification data is shown in Supplementary file 1.

Park H.J., Gregory M.A., Zaberezhnyy V., Goodspeed A., Jordan C.T., Kieft J.S, & DeGregori J. (2022). Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation. eLife, 11, e79940.

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Small Molecule Compounds in Cancer Research

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Doxorubicin was purchased from Cell Signaling Technology and dissolved in DMSO at 10mM; DL-α-Difluoromethylornithine (hydrochloride hydrate) was purchased from Cayman Chemical and dissolved in DMSO at 50mM; spermidine, thymidine, and aminoguanidine hydrochloride were purchased from Sigma and dissolved in water at 10mM,100mM or 1M, respectively; putrescine (dihydrochloride) was purchased from Santa Cruz Biotechnology and dissolved in water at 10mM; N-ω-Hydroxy-L-norarginine acetate salt was purchased from Bachem and dissolved in water at 50mg/mL. Cisplatin was obtained from the Dana Farber Cancer Institute pharmacy at 1mg/mL in PBS. DMSO for use as an organic solvent and vehicle control was purchased from Fisher Scientific. Antibodies ODC (MABS36, 1:100) was purchased from Millipore. ARG2 (ab137069, 1:1000) was purchased from Abcam. pH2A.X S139 (9718, 1:1000), beta-actin (4970, 1:1000), c-Myc (5605, 1:1000), and vinculin (13901, 1:1000) were purchased from Cell Signaling Technology. Antibodies were used at indicated dilutions in 5% milk (Andwin Scientific) in TBST buffer (Boston Bioproducts), except for p-H2A.X in 5% BSA (Boston Bioproducts) in TBST.

Geck R.C., Foley J.R., Stewart T.M., Asara J.M., Casero R.A., & Toker A. (2020). Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy.

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