P h2a x s139

Manufactured by Merck Group

P-H2A.X (S139) is a laboratory equipment product used to detect and quantify DNA double-strand breaks in cells. It measures the phosphorylation of the histone variant H2A.X at serine 139, which is a sensitive marker of DNA damage response.

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4 protocols using p h2a x s139

Immunoblot and Immunohistochemistry Analyses

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Antibodies and concentrations used: pThr18/Ser19-MLC2 (#3674; 1:750, immunoblot), pSer19-MLC2 (#3671; 1:50, immunohistochemistry; 1:200, immunofluorescence), MLC2 (#3672; 1:750), pT202/Y204-p44/42 (ERK1/2) (#4370; 1:1,000), pY705-STAT3 (#9145; 1:750), PD-L1 (clone E1L3N, #13684, 1:200) from Cell Signaling Technology; STAT3 (sc-482; 1:500), ERK2 (sc-154; 1:1,000), MCL-1 (sc-819; 1:1,000), GFP (sc-8334; 1:1,000) from Santa Cruz Biotechnology; GAPDH (MAB374; 1:10,000) from Millipore; P-H2A.X (S139) (ab2893;1:1000), CD206 (ab64693; 1:1,000), CD3 (anti-mouse, ab134096; 1:500), CD4 (anti-mouse, clone I3T4, ab183685; 1:300), FoxP3 (anti-human, clone 236A/E7, ab20034; 1:200) from Abcam; F4/80 (anti-mouse, clone BM8, MF48000, 1:1000), CD8a (anti-mouse, clone Ly2, 14-0808-82; 1:200), FoxP3 (anti-mouse, clone FJK-16s, 14-5773-82; 1:200) from Invitrogen; CD4 (anti-human, clone 11E9, NCL-L-CD4-368; 1:300) from Novocastra.

Orgaz J.L., Crosas-Molist E., Sadok A., Perdrix-Rosell A., Maiques O., Rodriguez-Hernandez I., Monger J., Mele S., Georgouli M., Bridgeman V., Karagiannis P., Lee R., Pandya P., Boehme L., Wallberg F., Tape C., Karagiannis S.N., Malanchi I, & Sanz-Moreno V. (2020). Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance. Cancer Cell, 37(1), 85-103.e9.

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Immunoblotting Antibody Validation

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Primary antibodies used for immunoblotting, immunofluorescence and immunohistochemistry were actin (Sigma), cleaved caspase 3 (Cell Signaling), CREB binding protein (CBP; Santa Cruz), cyclin A (Novocastra), pH2AX S139 (Millipore), H2AX (Bethyl), phospho-histone H3 S10, phospho-KIT Y719 (both Cell Signaling), Ki-67 (Thermo Scientific Neomarkers), KIT (Dako/Agilent), p27^Kip1 (Fisher/BD Biosciences Pharmingen), PARP (Invitrogen), cleaved PARP (Abcam) and mono-ubiquitin (BD Pharmingen).

Rausch J.L., Ali A.A., Lee D.M., Gebreyohannes Y.K., Mehalek K.R., Agha A., Patil S.S., Tolstov Y., Wellens J., Dhillon H.S., Makielski K.R., Debiec-Rychter M., Schöffski P., Wozniak A, & Duensing A. (2020). Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells. Scientific Reports, 10, 5178.

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Immunoblotting and FACS Analysis

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Immunoblotting was performed using the following antibodies diluted in TBS supplemented with 0.1% Tween 20 and 5% milk powder or 3% BSA: cyclin E1 (1:1000, Santa Cruz, sc-247), beta-actin (1:1000, Santa Cruz, sc-81178), p-p53 S15 (1:1000, Cell Signalling, 9284), p-CHK1 S345 (1:1000, Cell Signalling, 2348), p-RPA S4/S8 (1:5000, Bethyl Laboratories, A300-245), HA.11 (1:1000, BioLegend, 16B12), p53 (1:1000, Santa Cruz, sc-126), p21 (1:1000, Cell Signalling, 2947), HA (1:1000, Santa Cruz, sc-805), alpha-Tubulin (1:4000, Sigma, T5168), POT1 (1:1000, Novus Biologicals, NB500-176), FBXW7 (1:1000, Proteintech, 55290-1-AP), p-CDK1 T14/Y15 (in-house by Julian Gannon), Cdh1 (1:1000, in-house by Julian Gannon, AR38.2), anti-Mouse HRP (1:5000, Dako, P0447) and anti-Rabbit HRP (1:5000, Jackson Immuno, 711-035-152). The following antibodies were used for FACS and diluted in PBS supplemented with 1% BSA: MCM7 (1:200, Santa Cruz, sc-56324), cyclin B1 (1:200, Abcam, ab32053), p-H2A.X S139 (1:200, Millipore, 05-636), anti-Rabbit Alexa Fluor 555 (1:500, Thermo, A21428) and anti-Mouse Alexa Fluor 555 (1:500, Thermo, A21424).

Zeng J., Hills S.A., Ozono E, & Diffley J.F.X. (2023). Cyclin E-induced replicative stress drives p53-dependent whole-genome duplication. Cell, 186(3).

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Western Blot Analysis of Protein Expressions

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Proteins were isolated and their differential expressions were analyzed by Western blot, as described previously [21 (link),22 (link)]. Briefly, the cells were lysed using RIPA buffer (50 mM Tris HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100 or NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 10 mM NaF freshly supplemented with protease and phosphatase inhibitors). Protein concentrations were quantified using a Bradford assay (Bio-Rad), and the samples were normalized for equal loading. The samples were then heated to 100 °C for 10 min in the presence of 6× Laemmli buffer (Boston BioProducts; Milford, MA, USA). The proteins were resolved by SDS-PAGE and their expression was analyzed following immunoblotting using specific antibodies. The following antibodies were used in this study: FANCD2 (R&D MAB93691), pCHK2-T68 (Invitrogen PA5-104715), pRPA 32-S33 (Cell Signaling 10148s), pCHK1-S296 (Cell Signaling 90178s), pH2AX-S139 (Millipore 05-636), GAPDH (Santa Cruz 32233), and Vinculin (Cell Signaling 13901S).

Okechukwu C.C., Ma X., Sah N., Mani C., Palle K, & Gmeiner W.H. (2024). Enhanced Therapeutic Efficacy of the Nanoscale Fluoropyrimidine Polymer CF10 in a Rat Colorectal Cancer Liver Metastasis Model. Cancers, 16(7), 1360.

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