C myc d84c12

Manufactured by Cell Signaling Technology

Sourced in United States

C-Myc (D84C12) is a rabbit monoclonal antibody that recognizes the c-Myc protein. C-Myc is a transcription factor that plays a crucial role in cell proliferation, growth, and apoptosis. This antibody can be used for the detection of c-Myc in various experimental applications.

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C-Myc (671 citations) MYC (D84C12) (3 citations) C-Myc (D84C12,5605), (2 citations) C-Myc (D84C12) Rabbit mAb (2 citations) Anti-human c-Myc rabbit monoclonal (D84C12), (2 citations)

10 protocols using c myc d84c12

Protein Expression Analysis of Stimulated B Cells

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Freshly isolated B cells were cultured in complete RPMI 1640 with or without stimuli. After 24 hours, cells were collected and lysed with RIPA buffer (PBS, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 10 mM EDTA) plus 10 mM sodium fluoride and complete protease inhibitor cocktail (Roche). Protein content of cleared lysates was measured using the BCA Protein Assay kit (Thermo Fisher Scientific). Lysates were resolved on 4–12% or 10% polyacrylamide Bis-Tris gel (Bio-Rad or Invitrogen) and transferred onto PVDF membrane (EMD Millipore). The membrane was probed for the indicated proteins. The following antibodies were purchased from Cell Signaling: anti-MALT1, -Bcl-2 (D17C4), -β-actin (13E5), -phospho-Rb (Ser807/811) (D20B12), -cyclin D3 (DCS22), -c-Myc (D84C12), -survivin (71G4B7), and -total Akt. Anti-Mcl-1 was purchased from Rockland Immunochemicals, anti-cyclin D2 (M-20) from Santa Cruz, and anti-Bcl-xL from BD Biosciences.
Primary antibodies were then detected with horseradish peroxidase-labeled donkey anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch) and developed with the SuperSignal West Pico chemiluminescence kit (Thermo Fisher Scientific).

Lee P., Zhu Z., Hachmann J., Nojima T., Kitamura D., Salvesen G, & Rickert R.C. (2016). Differing requirements for MALT1 function in peripheral B cell survival and differentiation. Journal of immunology (Baltimore, Md. : 1950), 198(3), 1066-1080.

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Apoptosis and Cell Cycle Regulation

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PPI was purchased from the Institute for Drug Control (Shanghai, China, #111590) and dissolved in dimethyl sulfoxide (DMSO). Doxorubicin was purchased from Sangon Biotech (Shanghai, China, #DB3456) and dissolved in ultra-purified water. FITC Annexin V and propidium iodide were obtained from BD Biosciences (Franklin Lakes, USA, #556420). The cell cycle detection kit was from Key GENBioTECH (Nanjing, China). Antibodies against PARP (#9542), BAX (D2E11, #5023), BCL-2 (#2876), Vimentin (5G3F10, #3390), C-Myc (D84C12, #5605), GSK-3β (27C10, #9315), phosphor-GSK-3β (#8213), active β-catenin (D13A1, #8814) and β-catenin siRNA II (#6238) were from Cell Signaling Technology (Danvers, USA). Trypsin-EDTA, Lipofectamine 2000 (#11668) and TRIzol reagent (#15596) were from Invitrogen (Rockville, MD, USA). CHIR99021 (#SML1046) and antibody against β-actin (#A2228) were from Sigma-Aldrich (Saint Louis, USA). Monoclonal antibody of rabbit anti-Ki-67 and RIPA Cell Lysis Buffer (#P0013B) were got from Beyotime Institute of Biotechnology (Shanghai, China). SuperSignal Chemiluminescent HRP Substrate (#34078) was from Thermo Fisher scientific Inc (Rockford, USA). Matrigel (#356234) was purchased from BD Biosciences (Bedford, USA). PrimeScript RT reagent kit (#RR037A) and SYBR Premix Ex Taq II (#RR420L) were bought from TaKaRa (Dalian, China).

Chang J., Li Y., Wang X., Hu S., Wang H., Shi Q., Wang Y, & Yang Y. (2017). Polyphyllin I suppresses human osteosarcoma growth by inactivation of Wnt/β-catenin pathway in vitro and in vivo. Scientific Reports, 7, 7605.

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Comprehensive Immune Cell Profiling

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For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (R17217), and anti-CD98 (RL388; all from eBioscience). Intracellular Foxp3 (FJK-16s), Ki67 (SolA15), IFN-γ (XMG1.2), IL-4 (11B11), IL-17 (17B7; all from eBioscience), Bim (C34C5), c-Myc (D84C12), and p-S6 (D57.2.2E; all from Cell Signaling Technology) were analyzed by flow cytometry according to the manufacturer’s instructions. For intracellular cytokine staining, T cells were stimulated for 4 h with PMA plus ionomycin in the presence of monensin before intracellular staining according to the manufacturer’s instructions (eBioscience). Caspase-3 activity was measured using active caspase-3 apoptosis kit (BD Biosciences). To monitor cell division, lymphocytes were labeled with CellTrace^™ violet (Life Technologies). Flow cytometry data were acquired on LSRII or LSR Fortessa (BD Biosciences) and analyzed using Flowjo software (Tree Star).

Wei J., Long L., Yang K., Guy C., Shrestha S., Chen Z., Wu C., Vogel P., Neale G., Green D.R, & Chi H. (2016). Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nature immunology, 17(3), 277-285.

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Comprehensive Immune Cell Profiling

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Immunoblotting and Flow Cytometry Protocols

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Western blots, prepared by standard procedures, were probed using the following antibodies: c-Myc (N-262), Max (C-17) and Mnt (M-132) all from Santa Cruz Biotechnology (Dallas, TX, USA), or c-Myc (D84C12) from Cell Signaling Technology (Danvers, MA, USA) and Mnt (A303-626A) from Bethyl Laboratories (Montgomery, TX, USA) and β-actin (AC-74, Sigma-Aldrich, St. Louis, MO, USA). Blots in Figure 1b were imaged on a ChemiDoc Touch (Bio-Rad, Hercules, CA, USA) and analysed using Image Lab software (Bio-Rad). Monoclonal antibodies used for flow cytometry, produced and labelled with FITC, PE or APC in house, included the following: RB6-8C5, anti-Gr1; MI/70, anti-Mac1; YTA3.2.1, anti-CD4; 53.6.7.2, anti-CD8; Ter119, anti-erythroid marker; ID3, anti-CD19; RA3-6B2, anti-CD45R-B220; 5.1, anti-IgM; 11-26C, anti-IgD; 145-2C11, anti-CD3; E13.161.7, anti-Sca-1; 57, anti-CD43; T329.1, anti-Thy1; A20.1 anti-Ly5.1.

Campbell K.J., Vandenberg C.J., Anstee N.S., Hurlin P.J, & Cory S. (2017). Mnt modulates Myc-driven lymphomagenesis. Cell Death and Differentiation, 24(12), 2117-2126.

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Immunoblotting Analysis of Signaling Pathways

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The following antibodies were purchased from Cell Signaling Technology: HIF-1α (D1S7W) (Rabbit mAb#36,169); c-Myc (D84C12) (Rabbit mAb #5605); Phospho-Akt (Thr308) (244F9) (Rabbit mAb #4056); Phospho-Akt (Ser473) (587F11) (Mouse mAb #4051); Akt (pan) (11E7) (Rabbit mAb #4685); p70 S6 Kinase (49D7) (Rabbit mAb #2708); Phospho-p70 S6 Kinase (Thr421/Ser424) (Antibody #9204); Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (Rabbit mAb (HRP Conjugate) #8544); p44/42 MAPK (Erk1/2) (137F5) (Rabbit mAb #4695); FAK antibody ( #3285); Phospho-FAK (Tyr576/577) antibody (#3281); CDK4 (D9G3E) (Rabbit mAb #12790); Rb (4H1) (Mouse mAb #9309); Phospho-Rb (Ser807/811) (D20B12) (Rabbit mAb #8516); Cyclin D1 antibody (#2922), PRAS40 antibody (#2610); Phospho-PRAS40 (Thr246) (D4D2) (Rabbit mAb #13175). The followings reagents were purchased from ABCAM: Anti-LOX antibody (ab174316); Anti-TGF beta 1 antibody (ab27969); Anti-Ki67 antibody (ab15580). rLOX was purchased from OriGene Technologies. BAPN was purchased from Sigma-Aldrich.

Li Q., Zhu C.C., Ni B., Zhang Z.Z., Jiang S.H., Hu L.P., Wang X., Zhang X.X., Huang P.Q., Yang Q., Li J., Gu J.R., Xu J., Luo K.Q., Zhao G, & Zhang Z.G. (2019). Lysyl oxidase promotes liver metastasis of gastric cancer via facilitating the reciprocal interactions between tumor cells and cancer associated fibroblasts. EBioMedicine, 49, 157-171.

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Immunoblotting Analysis of PD-L1, β-catenin, and c-Myc

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Three million pCMV Vector or miR-200c transfected SKOV3 cells were collected and lysed in RIPA lysis buffer. Thirty micrograms of protein of each sample was loaded on 10% polyacrylamide gels and run for 1.5 h at 120 V. The following antibodies were used: PD-L1 (E1L3N) (Cell Signaling Technology, Danvers, MA, USA; Cat. n.13684), β-catenin (E-5) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany; Cat. n.sc-7963), and c-Myc (D84C12) (Cell signaling; Cat. n.5605). β-actin (C4) (Santa Cruz; Cat. n.sc-47778) and Lamin B1 (C-20) (Santa Cruz; Cat. n.sc-6216) were used to ensure equal protein loading. HRP conjugated anti-rabbit (SIGMA; Cat. n.A 6154) and anti-mouse (ADVANSTA; Cat. n.R-05071-500) secondary antibodies were used, (1:5000 in 2% BSA) for 30 min. The chemiluminescent signal was detected using WesternBright^® ECL (ADVANSTA, San Jose, CA, USA; Cat. n.K-12045-D20). Densitometry analysis was performed with ImageJ Software (v. 10.2). Immunoblots were repeated three times with the same lysates and with protein lysates derived from three different treatments of olaparib and irradiation. Additional details of immunoblotting conditions can be found in Supplementary Materials and Methods.

Anastasiadou E., Messina E., Sanavia T., Mundo L., Farinella F., Lazzi S., Megiorni F., Ceccarelli S., Pontecorvi P., Marampon F., Di Gioia C.R., Perniola G., Panici P.B., Leoncini L., Trivedi P., Lenzi A, & Marchese C. (2021). MiR-200c-3p Contrasts PD-L1 Induction by Combinatorial Therapies and Slows Proliferation of Epithelial Ovarian Cancer through Downregulation of β-Catenin and c-Myc. Cells, 10(3), 519.

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Signaling Pathways Modulation in Cancer

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Sorafenib (CAS #475207-59-1) and quizartinib (AC220, CAS #950769-58-1) were purchased from Selleck. Chloroquince (CQ, CAS #50-63-5) was bought from Sigma. The antibodies against human-phosphorylated (p)-p44/42 MAPK (ERK1/2, Thr202/Tyr204, CAS #4370), p-FLT3 (Tyr589/591, CAS ##60413), p-mTOR (Ser2448, CAS #5536), p-S6K (Ser240/244, CAS #2215), mTOR (CAS ##2983), S6K (CAS #9202), Beclin-1 (CAS #4122), LC3B (CAS #3868), ATG5 (D5G3, CAS #9980), p62/SQSTM1 (CAS #5114), c-Myc (D84C12, CAS #5605) and cleaved caspase-3 (Asp175, CAS #9661) were purchased from Cell Signaling Technology. Against ERK2 (CAS #sc-1647) and FLT3 (CAS #sc-19635) were from Santa Cruz Biotechnology. Anti-GAPHD (CAS #G9545) was purchased from Millipore Sigma.

Xu D., Chen Y., Yang Y., Yin Z., Huang C., Wang Q., Jiang L., Jiang X., Yin C., Liu Q, & Yu G. (2022). Autophagy activation mediates resistance to FLT3 inhibitors in acute myeloid leukemia with FLT3-ITD mutation. Journal of Translational Medicine, 20, 300.

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OTUB1-Mediated Apoptosis Regulation

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The following antibodies were obtained from Cell Signaling (Boston, MA, USA): c-Myc (D84C12, #12189); cleaved-caspase-3 (D3E9, #8172); cleaved-PARP (D64E10, #5625); cyclin D (92G2, #2978); E-cadherin (#3195); N-cadherin (D4R1H, #13116); MMP2 (D8N9Y, #13132); MMP9 (D6O3H, #13667); and GAPDH (D16H11, #5174). The following reagents were utilized: Lipofectamine 3000 transfection reagent (Lot. 11668-027 Invitrogen, Carlsbad, CA, USA) and RIPA lysis buffer (Lot. 89901, Thermo Scientific, USA). The pcDNA3.1-OTUB1-isoform 2 construct was constructed by Hanhen Co. Ltd. (Shanghai, China).

Wang Y.Q., Zhang Q.Y., Weng W.W., Wu Y., Yang Y.S., Shen C., Chen X.C., Wang L., Liu K.J., Xu M.D, & Sheng W.Q. (2016). Upregulation of the Non-Coding RNA OTUB1-isoform 2 Contributes to Gastric Cancer Cell Proliferation and Invasion and Predicts Poor Gastric Cancer Prognosis. International Journal of Biological Sciences, 12(5), 545-557.

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Protein Expression Analysis via Western Blot

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Cytoplasmic and nuclear extracts or whole cell lysates were prepared with NE-PER nuclear and cytoplasmic extraction reagents (ThermoFisher Scientific). Western blot analysis was performed as previously described [19 (link)]. The primary antibodies used were to KRT13 (EPR3671, Abcam, Cambridge, UK or B-2, Santa Cruz Biotechnology), CD44 (156-3C11, Cell Signaling, Danvers, MA), c-Myc (D84C12, Cell Signaling), ALDH1A1 (B-5, Santa Cruz Biotechnology), Nanog (5A10, Santa Cruz Biotechnology), PG (A-6, Santa Cruz Biotechnology), DSP [EPR4383(2), Abcam], Actin (AC-15, Santa Cruz Biotechnology) and Lamin B (C-20, Santa Cruz Biotechnology). Immunoblots were subjected to morphometric analysis by Image Studio Software (LI-COR, Lincoln, NE).

Yin L., Li Q., Mrdenovic S., Chu G.C., Wu B.J., Bu H., Duan P., Kim J., You S., Lewis M.S., Liang G., Wang R., Zhau H.E, & Chung L.W. (2022). KRT13 promotes stemness and drives metastasis in breast cancer through a plakoglobin/c-Myc signaling pathway. Breast Cancer Research : BCR, 24, 7.

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