C myc 9e10

Manufactured by Santa Cruz Biotechnology

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C-Myc (9E10) is an antibody that recognizes the c-Myc protein. The c-Myc protein is a transcription factor that plays a key role in cellular processes such as cell growth, proliferation, and apoptosis.

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C-Myc (386 citations) Myc (9E10) (31 citations) Anti-c-Myc (9E10, (25 citations) Mouse anti-c-Myc 9E10 (12 citations) C-Myc (clone 9E10; (11 citations) Anti-c-Myc (9E10; sc-40; (8 citations) Anti-c-Myc antibody (9E10, (7 citations) C-Myc Antibody (9E10), (6 citations) Mouse monoclonal anti-c-Myc (9E10) (5 citations) Mouse c-Myc (9E10) (4 citations)

39 protocols using c myc 9e10

Detailed Protocol for FOXP3 and TGF-β Signaling

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The primers used in this study were listed in Supplementary Table S6. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-flag-Smad2 (Addgen plasmid #14042) and CS2-flag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), p53 (DO-1, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Jia H., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B, & Chen G.G. (2020). Nuclear FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc. Oncogenesis, 9(10), 97.

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Molecular Pathways in Transcriptional Regulation

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The primers used in this study were listed in Supplementary Table S1. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-ag-Smad2 (Addgen plasmid #14042) and CS2-ag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Hao J., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B., & Chen G.G. (2020). FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc.

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Investigating Phosphorylation Signaling Pathways

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Antibodies against phospho-SHIP1 (Tyr1020), phospho-SHP-1 (Tyr564) (D11G5), phospho-Src family (Tyr416) (D49G4), phospho-p38 MAPK (Thr180/Tyr182) (3D7), phospho-(Ser) PKC substrate, phospho-p40^phox (Thr154), and Rac1/2/3 antibodies were purchased from Cell Signaling Technology. Phospho-ERK (E-4), p38α/β MAPK (H-147), p47^phox (H-195), p22^phox (C-17), Rab5 (D-11), Rab7 (H-50), calnexin (H-70), c-Myc (9E10), and COX-2 (C-20) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The iNOS/NOS type II (54/iNOS) antibody was purchased from BD Biosciences, and the β-actin (AC-15) antibody was purchased from Sigma-Aldrich. Secondary antibodies for Western blotting, ECL™ anti-rabbit, and anti-mouse IgG HRP were obtained from GE Healthcare. For immunofluorescence and confocal laser microscopy, Alexa Fluor^®488 goat anti-mouse IgG, Alexa Fluor^®488 goat anti-rabbit IgG, Alexa Fluor^®488 donkey anti-goat IgG, Alexa Fluor^®594 goat anti-rabbit IgG, and Alexa Fluor^®647 donkey anti-mouse IgG were obtained from Invitrogen. Phorbol 12-myristate 13-acetate (PMA) and pure lipopolysaccharide (LPS) from E. coli O111:B4 (L3024) were purchased from Sigma-Aldrich. The inhibitors, bisindolylmaleimide I (BIM-1), SB203580, and LY294992 were obtained from Calbiochem (San Diego, CA, USA).

Romero-Pinedo S., Barros D.I., Ruiz-Magaña M.J., Maganto-García E., Moreno de Lara L., Abadía-Molina F., Terhorst C, & Abadía-Molina A.C. (2022). SLAMF8 Downregulates Mouse Macrophage Microbicidal Mechanisms via PI3K Pathways. Frontiers in Immunology, 13, 910112.

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Isolation and Analysis of Cellular Fractions

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Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

Yi W., Gupta S., Ricker E., Manni M., Jessberger R., Chinenov Y., Molina H, & Pernis A.B. (2017). The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity. Nature Communications, 8, 254.

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Immunoblotting Procedure for GLUT1, c-Myc, HKII, and HSP90

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For immunoblotting, cells were washed twice with cold PBS and lysed in RIPA lysis buffer by incubating at 4°C rotatory shaker for 30 min. Cell debris was removed by centrifugation at 13,000 rpm for 10 min and the supernatant was collected. Protein content was measured by performing Bradford assay. Western blotting was performed as described previously [23 (link)]. The membranes were probed with primary antibody against GLUT1 (Abcam, Cambridge, UK), c-Myc-9E10 (Santa Cruz Biotechnology, Dallas, TX, USA), HKII (Cell Signaling Technology, Beverly, MA, USA), and HSP90 (Santa Cruz Biotechnology).

Shukla S.K., Gebregiworgis T., Purohit V., Chaika N.V., Gunda V., Radhakrishnan P., Mehla K., Pipinos I.I., Powers R., Yu F, & Singh P.K. (2014). Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia. Cancer & Metabolism, 2, 18.

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Multimodal Protein Analysis Protocol

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β‐catenin (610154, BD), caspase‐3 (9661L, Cell Signalling), Cyclin D1 (2978S, Cell Signalling), DVL2 (10B5) (sc‐8026, Santa Cruz), GAPDH (sc‐47724, Santa Cruz), Flag (F3165, SIGMA), HA (Y‐11) (sc‐7392, Santa Cruz), LGR4 (C‐12) (sc‐390630, Santa Cruz), Lysozyme (A0099, Dako), c‐Myc (9E10) (sc‐40, Santa Cruz), NEDD4 (sc‐25508, Santa Cruz), NEDD4L (4013S, Cell Signalling), SNAP (P9310S, NEB), Sox9 (AB5335, Millipore) and V5 (ab27671) were used in immunohistochemistry, immunoprecipitations or Western blot analysis.

Novellasdemunt L., Kucharska A., Jamieson C., Prange‐Barczynska M., Baulies A., Antas P., van der Vaart J., Gehart H., Maurice M.M, & Li V.S. (2019). NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor. The EMBO Journal, 39(3), e102771.

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Protein Immunoblot Analysis Protocol

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The protein samples were subjected to SDS‐PAGE. The proteins were then electrotransferred to PVDF membranes (Millipore) and subjected to immunoblot analysis. Antibodies against FLAG (M2; Sigma‐Aldrich), HA (3F10; Roche Diagnostics), c‐Myc (9E10; Santa Cruz Biotechnology), and phosphorylated tyrosine (4G10; Millipore) were used. The reacted Abs were detected as described previously.22

Xie R., Okita Y., Ichikawa Y., Fikry M.A., Huynh Dam K.T., Tran S.T, & Kato M. (2019). Role of the kringle‐like domain in glycoprotein NMB for its tumorigenic potential. Cancer Science, 110(7), 2237-2246.

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Chromatin Binding Assay Protocol

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Chromatin binding assay was performed as previously described with modifications [8] (link). Briefly, cells were cultured to an OD₆₀₀ of 0.4, arrested in pre-anaphase (nocodazole), pelleted and washed with 1.2 M Sorbitol. Cells were resuspended in CB1 buffer (50 mM Sodium citrate, 1.2 M Sorbitol, 40 mM EDTA, pH 7.4). Cells were spheroblasted, and resuspended in 1.2 M Sorbitol and frozen in liquid nitrogen. Cells were thawed on ice and supplemented with Lysis buffer (500 mM Lithium Acetate, 20 mM MgSO₄, 200 mM HEPES, pH 7.9), protease inhibitor cocktail (Sigma), and TritonX-100. Lysate was centrifuged at 12,000×g for 15 minutes and supernatant containing soluble fraction and pellet containing chromatin bound fraction were collected and supplemented with 4X Laemelli (Amresco). Whole cell extracts, supernatant, and pellet were resolved by SDS-PAGE and analyzed using c-Myc (9E10) (Santa Cruz), H2B (Santa Cruz), and PGK (Invitrogen).

Tong K, & Skibbens R.V. (2014). Cohesin without Cohesion: A Novel Role for Pds5 in Saccharomyces cerevisiae. PLoS ONE, 9(6), e100470.

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Western Blot Protein Detection

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Total protein extraction was performed as previously described (Barco et al., 2019b (link)). Extract [5 µl (DEX : MYB51-myc or DEX : WRKY33-flag) or 50 µl (DEX : MYB51-myc and DEX : WRKY33-flag)] was loaded onto a 10% SDS-PAGE gel, and the separated proteins were transferred to PVDF membrane (Millipore, Billerica, MA), stained with Ponceau S for labeling of total protein, and probed with either FLAG M2 (Sigma-Aldrich, cat# F1804) or c-Myc 9E10 (Santa Cruz Biotechnology, cat# sc-40) antibodies diluted 1:1,000 in 1X PBS containing 5% (w/v) nonfat milk.

Barco B, & Clay N.K. (2020). Hierarchical and Dynamic Regulation of Defense-Responsive Specialized Metabolism by WRKY and MYB Transcription Factors. Frontiers in Plant Science, 10, 1775.

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Western Blot Analysis of RBPs in MB Cells

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Protein lysates from MB primary cultures and frozen operative tissue were prepared in lysis buffer (50 mM HEPES, pH7.0, 150 mM NaCl, 2% SDS, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). Proteins were separated on 4–20% Bis-Tris gradient polyacrylamide gels (Invitrogen) and transferred onto Immuno-Blot nitrocellulose membranes (Bio-Rad Laboratories). Membranes were then incubated in blocking buffer (1X TBS containing 5% milk and 0.05% Tween-20, 1 hour), probed overnight with antibodies specific for HNRNPA2/B1 (Cell Signaling, 1:1000), HNRNPA1 (4B10; Santa Cruz; 1:2000), HNRNPC1/C2 (4F4; Santa Cruz; 1:5000), cMYC (9E10; Santa Cruz; 1:500) and actin (Sigma; 1:2000).

Staal J.A., Lau L.S., Zhang H., Ingram W.J., Hallahan A.R., Northcott P.A., Pfister S.M., Wechsler-Reya R.J., Rusert J.M., Taylor M.D., Cho Y.J., Packer R.J., Brown K.J, & Rood B.R. (2015). Proteomic profiling of high risk medulloblastoma reveals functional biology. Oncotarget, 6(16), 14584-14595.

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