Phospho c raf

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-c-Raf is an antibody that recognizes the phosphorylated form of the c-Raf protein. c-Raf is a serine/threonine protein kinase that plays a key role in the MAPK/ERK signaling pathway. The phosphorylation of c-Raf is a critical step in the activation of this pathway.

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Lab products found in correlation

Close spelling variants of this product

C-Raf (66 citations) Anti-phospho-c-Raf (7 citations) Phospho c-Raf (ser259) (4 citations) Phospho-Raf (3 citations)

21 protocols using phospho c raf

Synthesis and Evaluation of Small Molecule Inhibitors

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ISJ and its intermediate, compound 7, were synthesized in our laboratory. GF 109203X (GFX) and Go 6976 (GO) were purchased from Tocris Bioscience (Ellisville, MO). 5-Fluorouracil (5-Fu) was obtained from Sigma (St. Louis, Mo). Primary antibodies were purchased from Cell Signaling Technology (Beverly, MA), including those specific for β-actin (#4970), MMP-9 (#2270), E-Cadherin (#3195), FGFR-1 (#9740), PKCε (#2683), c-Raf (#9422), MEK1/2 (#9122), ERK1/2 (#9102), Phospho-c-Raf (#9427), Phospho-MEK1/2 (#9154), and Phospho-ERK1/2 (#4370). The PKC Isoform Antibody Sampler Kit (#9960) and the Apoptosis Antibody Sampler Kit (#9915) were also purchased from Cell Signaling Technology. Antibodies to p53 (AP062) and p21 (AP021) were purchased from Beyotime (Shanghai, China).

Yuan X., Chen H., Li X., Dai M., Zeng H., Shan L., Sun Q, & Zhang W. (2015). Inhibition of protein kinase C by isojacareubin suppresses hepatocellular carcinoma metastasis and induces apoptosis in vitro and in vivo. Scientific Reports, 5, 12889.

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Western Blot Analysis of Cellular Signaling

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Western blot analyses were performed as previously described [17 (link), 26 (link), 39 (link)]. Briefly, 90 % confluent NIH3T3 cells were serum-starved for 22 h. Cells were counted and lysed in 1× SDS running buffer, heated at 100 °C for 10 min, and centrifuged to remove debris. Lysates were resolved on 10 % polyacrylamide gels, transferred to nitrocellulose membranes, and blotted with the following primary antibodies: anti-KRAS4A specific antibody (1:500; Santa Cruz Biotechnology, sc-522), anti-RAS antibody (1:2000; Cell Signaling Technology, #3965), anti-actin (1:5000; Sigma-Aldrich, A1978), anti-GFP (1:2000; Cell Signaling Technology, #2555), and phospho-c-RAF, c-RAF, phoshpo-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-S6RP, and S6RP (All 1:2000; Cell Signaling Technology). HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG was used as a secondary antibody.

Zhao H., Liu P., Zhang R., Wu M., Li D., Zhao X., Zhang C., Jiao B., Chen B., Chen Z, & Ren R. (2015). Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A. Journal of Hematology & Oncology, 8, 132.

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Phosphoproteomic Profiling of Hepatocytes

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Proteins (10 μg) were separated by SDS-PAGE using 12% tris-glycine polyacrylamide gel electrophoresis and then transferred to a PVDF membrane using a wet blotting system (Roche, Basel, Switzerland) to profile phosphoproteins in hepatocytes. Membranes were blocked with 5% BSA in TBST (20 mM tris, 500 mM sodium chloride, 0.1% Tween-20, pH 7.5) for 4 h at room temperature (RT) and then incubated with primary antibodies at 4 °C for 18 h. The membranes were washed thrice with TBST for 10 min and then incubated with secondary antibodies for 1 h at RT. Signals were detected using iBright 1500 (Thermo Fisher Scientific) and ECL Prime Immunoblotting Detection Reagent (Cytiva, Marlborough, MA, USA).
To verify the phosphoproteomics results, primary antibodies specific to p53 (Cell Signaling Technology, Danvers, MA, USA; P/N 2524S), histone H3 (Cell Signaling Technology; P/N 9715S), α-tubulin (Abcam, UK; P/N ab52866), phospho-serine (Abcam, P/N ab9332), phospho-threonine (Cell Signaling Technology, P/N 9381S), phospho-tyrosine (Cell Signaling Technology, P/N 9411S), phospho-c-Raf (Cell Signaling Technology, P/N 9421S), phospho-MEK1/2 (Cell Signaling Technology, P/N 9154S), phospho-ERK1/2 (Cell Signaling Technology, P/N 9101S), and ERK1/2 (Cell Signaling Technology, P/N 9102S) were used.

Kim D., Lee M.S., Sung E., Lee S, & Lee H.S. (2022). Characterization of the RAS/RAF/ERK Signal Cascade as a Novel Regulating Factor in Alpha-Amanitin-Induced Cytotoxicity in Huh-7 Cells. International Journal of Molecular Sciences, 23(20), 12294.

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Amlodipine and Gefitinib Apoptosis Assay

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Amlodipine and gefitinib were purchased from Selleck Chemicals (Houston, TX, United States) and TopScience (Shanghai, China), respectively. Propidium iodide (PI) was obtained from Sigma-Aldrich (St. Louis, MO, United States). The Annexin V/FITC-PI apoptosis detection kit was obtained from BD Biosciences (San Jose, CA, United States of America). RPMI 1640 and FBS were purchased from Biological Industries (Beit Haemek, Israel). The enhanced chemiluminescence (ECL) reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Antibodies against phospho-EGFR, phospho-PDK1 (Ser241), PDK1, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-mammalian target of rapamycin complex 1 (mTORC1) (Ser2448), mTOR, phospho-glycogen synthase kinase 3 (GSK-3) β, phospho-p70 S6K (Thr389), p27, cyclin D1, phospho-retinoblastoma protein (Rb), phospho-extracellular signal-regulated kinase (ERK) 1/2, phospho-c-Raf, β-actin, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, United States). Antibody specific for p21 was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States).

Fu B., Dou X., Zou M., Lu H., Wang K., Liu Q., Liu Y., Wang W., Jin M, & Kong D. (2022). Anticancer Effects of Amlodipine Alone or in Combination With Gefitinib in Non-Small Cell Lung Cancer. Frontiers in Pharmacology, 13, 902305.

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Investigating MAPK Pathway Signaling

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Antibody of EGF Receptor (#2232), Phospho-BRAF (Ser445, #2696), CRAF (#53745), Phospho-c-Raf (Ser338, #9427), MEK1/2 (#4694), MEK1 (#12671), MEK1 (#2352), MEK2 (#9147), p-MEK1/2 (Ser217/221, #9154), ERK1/2 (#4695), p-ERK1/2 (Thr202/Tyr204, #4370), GST-Tag (#2624), PD-L1 (#13684) were obtained from Cell Signaling Technology (Danvers, MA), BRAF V600E (#A1137-25) from Biovision (San Francisco, USA), and BRAF (#ab33899), Ki67 (#ab16667) obtained from Abcam (Cambridge, USA). β-Actin (#sc47778) provided by Santa Cruz Biotechnology (Santa Cruz, USA), and anti-Flag antibody (#F1804) was obtained from Sigma–Aldrich (St. Louis, USA). FITC Rat Anti-Mouse CD45 (#553079), PerCP-Cy™5.5-CD8a (#551162), APC-NK-1.1 (#550627), PE-CD4 (#557308) obtained from BD (New Jersey, USA), and in vivo anti-mouse PD-L1 (#BE0101) obtained from BioXcel, USA.

Wang P., Jia X., Lu B., Huang H., Liu J., Liu X., Wu Q., Hu Y., Li P., Wei H., Liu T., Zhao D., Zhang L., Tian X., Jiang Y., Qiao Y., Nie W., Ma X., Bai R., Peng C., Dong Z, & Liu K. (2023). Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2. Signal Transduction and Targeted Therapy, 8, 96.

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Evaluating Protein Expression in Tissue Samples

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Lesional tissues were homogenized and cell samples were lysed in a buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄,1 μg ml ⁻¹ leupeptin and protease inhibitor cocktail). For detection of HIF-1a and SNAI1, cells were frozen with liquid nitrogen and then lysed in whole cell extraction buffer (10 mM HEPES (pH 7.9), 400 mM NaCl, 0.1 mM EDTA, 5% glycerol, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg ml⁻¹ leupeptin and protease inhibitor cocktail). Immuno-blotting was performed with primary antibodies against AKAP12 (Santa Cruz, 1:6,000), RARβ (Santa Cruz, 1:1,000), α-tubulin (InnoGenex, 1:8,000), E-cadherin (BD, 1:3,000), HIF-1α (BD, 1:3,000), ZO-1 (Invitrogen, 1:3,000), occludin (Invitrogen, 1:3,000), α-SMA (Dako, 1:1,000), SNAI1 (Cell Signaling, 1:500), pan-phospho-PKC (Cell Signaling, 1:1,000), phospho-C-raf (Cell Signaling, 1:1,000), C-raf (Cell Signaling, 1:3,000), phospho-MEK1 (Cell Signaling, 1:1,000), MEK1 (Cell Signaling, 1:3,000), phospho-p38 (Cell Signaling, 1:1,000), p38 (Cell Signaling, 1:3,000), phospho-Smad2 (Cell Signaling, 1:1,000), phospho-Smad3 (Cell Signaling, 1:1,000) and Smad2/3 (Cell Signaling, 1:6,000). The images of the full blots are presented in Supplementary Fig. 6.

Cha J.H., Wee H.J., Seo J.H., Ahn B.J., Park J.H., Yang J.M., Lee S.W., Lee O.H., Lee H.J., Gelman I.H., Arai K., Lo E.H, & Kim K.W. (2014). Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury. Nature communications, 5, 4952.

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Molecular Signaling Pathway Characterization

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Tan IIA was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), Go 6983, tanespimycin (17-AAG) and ganetespib from Medchem Express (Monmouth Junction, NJ, USA). Antibodies specific for β-actin (#4970), Hsp70 (#4872), IKKα (#2682), EGFR (#4267), PKCα (#2056), PKCδ (#9616), PKCμ (#2052), PKCζ (#9368), PKCε (#2683), Phospho-c-Raf (#9427), c-Raf (#9422), Phospho-MEK1/2 (#9154), MEK1/2 (#8727), Phospho-Erk1/2 (#4370), Erk1/2 (#9102), Phospho-PI3K (#4228), PI3K (#4257), Phospho-Akt (#4060), Akt (#9272), Phospho-mTOR (S2448) (#5536), Phospho-mTOR (S2481) (#2974), mTOR (#2983), LC3B (#3868), Bcl-2 (#2872), PARP (#9542) and cleaved caspase-3 (#9661) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Hsp90 (#ab13492) and anti-Ras (#ab52939) antibodies were from Abcam (Cambridge, UK).

Lv C., Zeng H.W., Wang J.X., Yuan X., Zhang C., Fang T., Yang P.M., Wu T., Zhou Y.D., Nagle D.G, & Zhang W.D. (2018). The antitumor natural product tanshinone IIA inhibits protein kinase C and acts synergistically with 17-AAG. Cell Death & Disease, 9(2), 165.

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Antibody Panel for HCRP-1 and MAPK Signaling

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Anti-HCRP-1 rabbit polyclonal antibodies were purchased from proteintech (Wuhan, China). Rabbit monoclonal antibodies to MMP-2, MMP-9, p38, phospho-p38, C-Jun amino terminal kinase (JNK), phospho-JNK, MEK1/2, phospho-MEK1/2, c-Raf, phospho-c-Raf, Ras and phospho-Ras were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against EGFR, phospho-EGFR (Tyr1173), ERK and phospho-ERK were from Santa Cruze (CA, USA). Mouse anti-β-actin was from Boster Biotechnology (Wuhan, China). ERK1/2 inhibitor, PD98059 and EGFR inhibitor, AG1478 were from Abcam (Shanghai, China).

Chen F., Deng J., Liu X., Li W, & Zheng J. (2015). HCRP-1 regulates cell migration and invasion via EGFR-ERK mediated up-regulation of MMP-2 with prognostic significance in human renal cell carcinoma. Scientific Reports, 5, 13470.

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Western Blot Analysis of EGFR Signaling

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Cells were lysed in RIPA buffer (Tris pH 7.4 50 mM, NaCl 150 mM, NP-40 1%, SDS 0.1%, EDTA 2 μM) containing proteinase inhibitors (Roche) and phosphatase inhibitors (Roche). The cell lysates (20 μg protein) were subjected to SDS-PAGE and Western blot. Antibodies against the following proteins were used: phospho-EGFR (Y1068), phospho-EGFR (Y845), EGFR, phospho-MEK1/2 (S217/221), MEK1/2, phospho-ERK (T202/Y204), ERK, phospho-AKT (S473), AKT, ERBB3, INSR, DUSP4, DUSP6, Rab11, E-Cadherin, PTEN, AXL, HER2, CRKL, MET, IGF-1R, ARAF, BRAF, phospho-CRAF (S338), CRAF, NRAS, and Actin (Cell Signaling Technology).

Ma P., Fu Y., Chen M., Jing Y., Wu J., Li K., Shen Y., Gao J.X., Wang M., Zhao X, & Zhuang G. (2016). Adaptive and Acquired Resistance to EGFR Inhibitors Converge on the MAPK Pathway. Theranostics, 6(8), 1232-1243.

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Kinase Inhibitor Evaluation Protocol

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Lapatinib, OSI-906, jnj38877605, MK2206, and U0126 were purchased from Selleck Chemicals. LY294002 and triciribine were purchased from Enzo Life Sciences. GSK2334470 was purchased from MedChem Express. The following antibodies were purchased from Cell Signaling Technology: phospho-EGFR (#3777), phospho-InsR/IGF1R (#3024), phospho-Met (#3077), phospho-Akt (#4060), phospho-Erk1/2 (#9101), phospho-CRAF (#9427), phospho-IRS1 (#3070) and IRS1 (#2382). phospho-IRS1 (pY632) antibody (ab109543) and vinculin antibody (ab73412) were purchased from Abcam. GAPDH antibody was purchased from Shanghai Kangchen. α-tubulin antibody was purchased from Santa Cruz. And β-actin antibody was purchased from Abmart.

Song Q., Sun X., Guo H, & Yu Q. (2016). Concomitant inhibition of receptor tyrosine kinases and downstream AKT synergistically inhibited growth of KRAS/BRAF mutant colorectal cancer cells. Oncotarget, 8(3), 5003-5015.

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