P h3s10

Manufactured by Cell Signaling Technology

Sourced in United States

P-H3S10 is a monoclonal antibody that detects phosphorylation of histone H3 at serine 10. It is used to analyze cell proliferation and mitotic progression.

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Lab products found in correlation

11 protocols using p h3s10

Immunohistochemical Analysis of Liver Tissues

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Liver specimens were fixed in 10% neutral buffered formalin for 36–48 hours. Paraffin sections (4 μm thick) were prepared and performed a series of dewaxing, rehydration, antigen retrieval, and quenching of endogenous peroxidase activity. The sections were incubated with the corresponding primary antibody at 4°C overnight and anti-mouse/rabbit secondary antibody (Dako REAL EnVision Detection System, Glostrup, Denmark) for 1 hour at room temperature. Detection was developed using the 3,3′-diaminobenzidine substrate. Primary antibodies were cleaved caspase 3 (cat. 9664; Cell Signaling Technology), pH3S10 (cat. 06-570; Merck Millipore, Burlington, MA), BrdU (cat. MS-1058-P0; Thermo Fisher Scientific, Waltham, MA), and Ki67 (cat. RM-9106-S1; Thermo Fisher Scientific).

Cao X., Shu Y., Chen Y., Xu Q., Guo G., Wu Z., Shao M., Zhou Y., Chen M., Gong Y., Li C., Shi Y, & Bu H. (2021). Mettl14-Mediated m6A Modification Facilitates Liver Regeneration by Maintaining Endoplasmic Reticulum Homeostasis. Cellular and Molecular Gastroenterology and Hepatology, 12(2), 633-651.

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Synthesis and Characterization of Anticancer Agents

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MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).

Tu H.J., Lin Y.J., Chao M.W., Sung T.Y., Wu Y.W., Chen Y.Y., Lin M.H., Liou J.P., Pan S.L, & Yang C.R. (2018). The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells. Clinical Epigenetics, 10, 162.

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Comprehensive Protein Detection Assay

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Primary antibodies raised against the following proteins were used for Western blotting: Rad51, Mdm2, Cyclin A2, pCdk Tyr 15, and H2AX (Abcam, Cambridge, MA, USA), beta-actin (Gene Script, Piscataway, NJ, USA), pH2AX S139, PARP, Rad51 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pH3 S10, p53 S15, total and cleaved forms of PARP, caspase-3, Chk1, Chk2, pChk1 S 345, pChk2, and Thr68 (Cell Signaling, Danvers, MA, USA) and PARP (Invitrogen, Carlsbad, CA, USA). HRP-conjugated secondary antibodies for Western blotting were purchased from Santa Cruz Biotechnology. For immunofluorescence staining, Cy3-labeled (Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor-488-labeled and 647-labeled secondary antibodies (Invitrogen) were used.

Boichuk S., Galembikova A., Bikinieva F., Dunaev P., Aukhadieva A., Syuzov K., Zykova S., Igidov N., Ksenofontov A, & Bocharov P. (2021). 2-APCAs, the Novel Microtubule Targeting Agents Active against Distinct Cancer Cell Lines. Molecules, 26(3), 616.

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Metformin and Salicylate Signaling Pathway

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Metformin, Salicylate and all standard chemicals were obtained from Sigma Aldrich. All antibodies (AMPK, p-AMPK T-175, ACC, p-ACC-S-79, H3 and P-H3-S10, HIF1a, p70^S6k, P-p70^S6k-T389, P-S6-S235/236, P-4EBP1-T37/46 and β-actin) were purchased from (Cell Signaling Technology). Biotinylated goat-anti-rabbit secondary antibody conjugated with streptavidin peroxidase and Nova Red were from Vector labs (Burlingame, CA).

Tsakiridis E.E., Broadfield L., Marcinko K., Biziotis O.D., Ali A., Mekhaeil B., Ahmadi E., Singh K., Mesci A., Zacharidis P.G., Anagnostopoulos A.E., Berg T., Muti P., Steinberg G.R, & Tsakiridis T. (2021). Combined metformin-salicylate treatment provides improved anti-tumor activity and enhanced radiotherapy response in prostate cancer; drug synergy at clinically relevant doses. Translational Oncology, 14(11), 101209.

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Western Blot Analysis of AgNPs Effect

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Cells treated with AgNPs or AgNO₃ were lysed in lysis buffer and Western blotting was performed as described previously [15 (link)]. Primary antibodies against p-H3S10, γ-H2AX, phospho-ERK, ERK, phosphor-JNK, JNK, phosphor-p38, p38 (Cell Signaling Technology Inc., Danvers, MA, USA) (1:1000) were used, followed by secondary antibodies conjugated with horseradish peroxidase (Jackson Immuno Research Laboratories, West Grove, PA, USA) (1:1000).

Zhao X., Rao Y., Liang J., Lin S., Wang X., Li Z, & Huang J. (2019). Silver Nanoparticle-Induced Phosphorylation of Histone H3 at Serine 10 Involves MAPK Pathways. Biomolecules, 9(2), 78.

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Protein Expression Analysis in Cellular Extracts

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Cell extracts (30 μg) were prepared in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with a protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN) and separated on 12% SDS–PAGE gels. The expression level of the indicated proteins was assessed by probing with the following antibodies: Tfcp2l1 (OAAB09732; Aviva Systems Biology), phosphorylated Tfcp2l1 (p‐Tfcp2l1; home‐made; AbFrontier), phosphorylated threonine (p‐Thr; #9381; Cell Signaling Technology), Flag‐epitope (F3165; Sigma‐Aldrich), HA epitope (G046; Abcam), Oct‐4 (sc‐5279; Santa Cruz), Nanog (ab14959; Abcam), SOX‐2 (2683‐S; Epitomics), Klf2 (09‐820; Millipore), Klf4 (#4038; Cell Signaling Technology), cyclin A (sc‐751; Santa Cruz), cyclin B (sc‐752; Santa Cruz), cyclin D (sc‐758; Santa Cruz), cyclin E (sc‐481; Santa Cruz), CDK1 (sc‐54; Santa Cruz), histone H3 phosphorylated at serine‐10 (p‐H3S10; #9701S; Cell Signaling Technology), Hdac1 (sc‐7872; Santa Cruz), Hdac2 (sc‐7899; Santa Cruz), Hdac3 (sc‐11417; Santa Cruz), Mta1 (#5646S; Cell Signaling Technology), Mbd3 (#3896S; Cell Signaling), Ruvbl2 (#12668S; Cell Signaling Technology), Tip60 (sc‐25378; Santa Cruz), DNMAP‐1 (10411‐1‐AP; Proteintech), Trrap (#3966S; Cell Signaling Technology), pCAF (sc‐8999; Santa Cruz), and β‐actin (A5441; Sigma‐Aldrich).

Heo J., Noh B., Lee S., Lee H., Kim Y., Lim J., Ju H., Yu H.Y., Ryu C., Lee P.C., Jeong H., Oh Y., Kim K., Kim S., Son J., Hong B., Kim J.S., Cho Y.M, & Shin D. (2019). Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis. EMBO Molecular Medicine, 12(1), e10880.

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Antibody Sources for Cell Signaling

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Rabbit polyclonal antibodies for BubR1 were developed in the laboratory. Antibodies for Myc-Tag, p-H3S10, Cyclin B1, and β-actin were purchased from Cell Signaling Technology Inc. Mouse monoclonal antibody for Mps1 was purchased from Invitrogen. GFP antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-FLAG and anti-α-Tubulin were purchased from Sigma Aldrich. Mouse anti-SUMO-1 and SUMO-2/3 antibodies were kindly provided by Dr. Michael J. Matunis (John Hopkins University). Human IgGs (CREST) against centromere proteins were purchased from Antibodies Incorporated (Davis, CA).

Restuccia A., Yang F., Chen C., Lu L, & Dai W. (2015). Mps1 is SUMO-modified during the cell cycle. Oncotarget, 7(3), 3158-3170.

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Cell Cycle Regulation Signaling Pathway

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Cell lysates were fractionated on SDS-PAGE gel and transferred to PVDF membrane. Membrane was blocked with 5% milk and incubated with primary antibody. Antibodies used were cleaved caspase-3 (Cell Signaling Technology), cleaved PARP (Cell Signaling Technology), p21(Cell Signaling Technology), p53 (BioLegend), GAPDH (Cell Signaling Technology), β-TUBULIN (Cell Signaling Technology), pCDC2(Y15) (Genewiz), Cyclin-B (Cell Signaling Technology), pCHK1(S317) (Cell Signaling Technology), pCHK1(S345) (Cell Signaling Technology), pCHK2(T68) (Cell Signaling Technology), pH3(S10) (Cell Signaling Technology), pH3(S28) (Cell Signaling Technology).

Negi S.S, & Brown P. (2015). rRNA synthesis inhibitor, CX-5461, activates ATM/ATR pathway in acute lymphoblastic leukemia, arrests cells in G2 phase and induces apoptosis. Oncotarget, 6(20), 18094-18104.

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KRCC1 Silencing and Cell Proliferation

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HeLa cells growing on coverslips were transfected with scrambled (siCTL) or KRCC1 siRNA (siKRCC1). Cells were pulse labeled with 5-ethynyl-2′-deoxyuridine (EdU) for 15 min, fixed and followed by immunofluorescence of pH3-S10 (Cell Signaling Technology, 1:300) and click reaction for EdU. Statistical analysis was performed using Student’s t test (n = 3). Differences were considered significant at P < 0.05.

Neizer-Ashun F., Dwivedi S.K., Dey A., Thavathiru E., Berry W.L., Lees-Miller S.P., Mukherjee P, & Bhattacharya R. (2022). KRCC1, a modulator of the DNA damage response. Nucleic Acids Research, 50(19), 11028-11039.

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Optimized Immunofluorescence Assay for DNA Damage Response

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Primary: CTC1 (33), STN1 (Abcam, 119263), Actinin (Santa Cruz, sc17829), a-Tubulin (Sigma, T-9026), CHK1 (Bethyl, A300-298A), CHK1 S317 (Bethyl, A304-673A), p53 (Cell Signaling, 9282), p21 (Santa Cruz, sc-6246), H3 (Cell Signaling, 9715), pH3 S10 (Cell Signaling, 9706), Rad9 (Santa Cruz, sc-74464), RPA32 (Abcam, ab16850), pRPA32 S33 (Bethyl, A300-246A), TopBP1 (Bethyl, A300-111A), ETAA1 (kindly provided by Dr. David Cortez), γH2AX (Bethyl, A300-081A) and POT1 (Abcam, ab124784).
Secondary: Thermo: anti-rabbit-HRP (32460); anti-mouse-HRP (32430); Molecular Probes: goat-antirabbit AlexaFluor 647 (A21244), goat-anti-mouse AlexaFluor 647 (A21235), goat-anti-mouse AlexaFluor 594 (A11032), goat-anti-rabbit AlexaFluor 594 (A11037). ATR inhibitor: VE-821 5 µM for 24 h (Selleckchem, S8007).

Ackerson S.M., Gable C.I., & Stewart J.A. (2020). Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress.

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