Anti ras

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-Ras is a laboratory reagent produced by Cell Signaling Technology. It is a tool used in research to detect and study the Ras protein family, which are key regulators of cell growth and differentiation.

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17 protocols using anti ras

Cytotoxic Effects of C086 on NSCLC

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The human NSCLC cell lines A549 and NCI-H1975 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 media containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO₂.
The bacterial strains and plasmids were obtained from the School of Life Science of Xiamen University, China. C086 (purity≥99%) was designed and synthesized by our laboratory (Figure 1B). C086 was dissolved in DMSO as a stock solution and diluted in culture media. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA). Anti-Hsp90, anti-β-actin, anti-EGFR, anti-Ras, anti-C-Raf, anti-Akt, anti-P-Akt, anti-Mek, anti-P-Mek, anti-Erk½, anti-P-Erk, anti-C-Myc anti-Bax, anti-Bcl-2 (an apoptosis suppression protein), caspase-8, cleaved caspase-8, and the Apoptosis Antibody Sampler Kit containing PARP, cleaved PARP, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7 were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Annexin-V-APC/PI Apoptosis Detection Kit was purchased from Nanjing Keygen Biotech Co. Ltd (Nanjing, China). Propidium iodide (PI) was obtained from Sigma Aldrich.

Wang L., Fan Y., Mei H., Liu Y., Zhang L., Xu J, & Huang X. (2019). Novel Hsp90 Inhibitor C086 Potently Inhibits Non-Small Cell Lung Cancer Cells As A Single Agent Or In Combination With Gefitinib. Cancer Management and Research, 11, 8937-8945.

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Western Blotting Analysis of Signaling Pathways

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Whole‐cell lysates were prepared in M‐PER buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), resolved by SDS/PAGE, and blotted with indicated antibodies. SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) were used to enhance blotting signal when needed. The following antibodies were used in this study: anti‐MAP4K4, anti‐phospho‐ERK1/2, anti‐phospho‐JNK, anti‐JNK, anti‐phospho‐p38, anti‐p38, anti‐AKT, anti‐phospho‐AKT, anti‐MEK1/2, anti‐phospho‐MEK1/2, anti‐RAS and anti‐PP2A‐C (Cell Signaling Technology, Danvers, MA, USA); anti‐ERK1/2, anti‐GAPDH, anti‐β‐actin, anti‐PP2A‐B56β, and anti‐MKP3 (Santa Cruz Biotechnology, Dallas, TX, USA); anti‐6Χ HIS (Immunology Consultants Laboratory, Inc., Lake Oswego, OR, USA). Erlotinib and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). The protein phosphatase 2 (PP2A) inhibitor okadaic acid (OA) was purchased from Sigma Aldrich. The PP2A activator FTY720 was purchased from Cayman Chemical (Ann Arbor, MI, USA). MAP4K4 inhibitor PF‐06260933 was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). EGF was obtained from Thermo Fisher Scientific.

Gao X., Chen G., Gao C., Zhang D.H., Kuan S., Stabile L.P., Liu G, & Hu J. (2017). MAP4K4 is a novel MAPK/ERK pathway regulator required for lung adenocarcinoma maintenance. Molecular Oncology, 11(6), 628-639.

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Protein Immunoprecipitation and Western Blotting

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Protein extracts and immunoprecipitation were resuspended in Laemmli 2X. SDS-PAGE and subsequent protein transfer to PVDF membrane, blocking and washes were performed at standard conditions. Primary antibodies utilized were anti-DBC1 (Bethyl, #A300-434 and #A300-432), anti-SIRT1 (Cell Signalling, #9475), anti-Cyclin D1 (Abcam, #ab137875), anti-β actin (Merck, #A5441), anti-α tubulin (Merck, #T6074), anti-GAPDH (Abcam, #ab9485 and Cell Signalling, #2118), anti-cyclin A2 (Abcam, #ab181591), anti-Ras (Cell Signalling, #3339) and anti-FLAG (Sigma, #F3165). Secondary antibodies utilized were HRP-conjugated IgG from rabbit, mouse and goat (Merck, #A0545, #A9044 and #A8919, respectively). Blot developing was performed with SuperSignal West Pico Chemiluminescent Kit (Pierce, #34080) and results were processed by densitometry analysis with ImageJ (Rasband, W.S., Bethesda, Maryland, USA).

Santos L., Colman L., Contreras P., Chini C.C., Carlomagno A., Leyva A., Bresque M., Marmisolle I., Quijano C., Durán R., Irigoín F., Prieto-Echagüe V., Vendelbo M.H., Sotelo-Silveira J.R., Chini E.N., Badano J.L., Calliari A.J, & Escande C. (2019). A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo. Scientific Reports, 9, 14381.

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Lung Tissue Protein Expression Analysis

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Mouse lung tissues and cell lines were homogenized in lysis buffer including RIPA buffer, protease and phosphatase inhibitor (Roche). Protein concentration was measured using Bradford Assay (Bio-Rad Laboratories). Western blot analysis was performed using 20 μg/lane protein according to standard procedures using polyvinylidene difluoride membranes and an enhanced chemiluminescence system (GE Healthcare). Anti-phospho STAT3 (Tyr705) (Clone D3A7) (Cell Signaling Technology, 1:2000), anti-phospho Akt (Ser473) (Cell Signaling Technology, 1:1000), anti-Akt (Cell Signaling Technology, 1:1000), anti-STAT3 (Cell Signaling Technology, 1:2000), anti-STAT1 (Cell Signaling Technology, 1:1000), anti-phospho ERK (Cell Signaling Technology, 1:2000), anti-ERK (Cell Signaling Technology, 1:1000), anti-Ras (Cell Signaling Technology, 1:1000), anti-Ras (G12D) (Cell Signaling Technology, 1:1000), anti-gp130 (CloneM-20) (Santa Cruz,1:200), anti-phospho c-Raf (Cell Signaling, 1;1000), anti-c-Raf (Cell Signaling, 1:1000), anti-Bcl-2 (Cell Signaling, 1:1000), anti-caspase-3 (Cell Signaling, 1:1000), anti-β-actin (Clone AC-15) (Sigma, 1:20000), and anti-β-tubulin (Cell Signaling, 1:2000) were used for Western blot analysis. A total of 20 mice, consisting of 5 of each combination of diet and genotype, were used for all analyses. Western blots were all repeated at least once.

Unver N., Delgado O., Zeleke K., Cumpian A., Tang X., Caetano M.S., Wang H., Katayama H., Yu H., Szabo E., Wistuba I., Moghaddam S.J., Hanash S, & Ostrin E.J. (2017). Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol. International journal of cancer, 142(7), 1405-1417.

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Molecular Mechanisms of Zoledronate-Induced Cell Death

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Anti-NF-κB, anti- Bcl-2, anti-phospho-Raf-1, anti-Akt, anti-vimentin, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti- cleaved PARP, anti-caspase-3, anti-Ras, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2, anti-p-ERK1/2, anti-p-Akt(Ser473), and anti-E-cadherin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-γ-H2A histone family member X antibodies were obtained from Millipore (Billerica, MA, USA). ZOL was purchased from Sigma–Aldrich (St. Louis, MO, USA). For in vitro experiments, ZOL was dissolved in PBS to prepare a 2 mM stock solution and stored at −20 °C.

Kim E.H., Kim M.S., Takahashi A., Suzuki M., Vares G., Uzawa A., Fujimori A., Ohno T, & Sai S. (2020). Carbon-Ion Beam Irradiation Alone or in Combination with Zoledronic acid Effectively Kills Osteosarcoma Cells. Cancers, 12(3), 698.

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Western Blot and Apoptosis Analysis

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Conditions for denaturing polyacrylamide gel electrophoresis (using a 10% separation gel) and Western blotting procedure were essentially as described before (Bartkova et al., 2008). Protein concentration was measured using the Bradford method and 15 μg of total proteins were resolved for each sample. The following antibodies were used for immunoblots, mouse anti‐beta‐Actin (A1978, Sigma, 1:1000), anti‐Ras (mouse anti‐HRas‐01, a gift from V. Horejsi, 1:100) and rabbit anti‐cMyc (5605, Cell Signaling, 1:1000).
Apoptotic cells were identified by propidium iodide exclusion from live cells stained with Hoechst 33342. Cells grown in 24‐well plates were stained for 10 min (RT) with each dye. Images were taken immediately for the red and blue channels respectively and processed using ImageJ software.

Maya-Mendoza A., Ostrakova J., Kosar M., Hall A., Duskova P., Mistrik M., Merchut-Maya J.M., Hodny Z., Bartkova J., Christensen C, & Bartek J. (2014). Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress. Molecular Oncology, 9(3), 601-616.

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Antibody panel for cell signaling study

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Anti PI3KC2α (#22028‐1‐AP, Proteintech), anti GFP (gift from Emilia Turco, University of Turin, Italy), anti α‐tubulin (#2125, Cell Signaling), anti GAPDH (sc‐47724, Santa Cruz Biotechnology), anti Myc‐tag (#2276, Cell Signaling), anti FAK (#71 433, Cell Signaling), anti p‐FAK (tyr397) (#8556, Cell Signaling), anti p‐FAK (tyr925) (#3284, Cell Signaling), anti Paxillin (#2542, Cell Signaling), anti p‐Paxillin (tyr118) (#69 363, Cell Signaling), anti HA‐tag (# 26 183, Thermofisher), anti R‐RAS (#8446, Cell Signaling), anti RASA3 (#PA5‐30445,Invitrogen), anti Rap1A/Rap1B (#4938, Cell Signaling), anti RAS (#3339, Cell Signaling), and anti Vinculin (#V9131, Sigma).

Li H., Prever L., Hsu M.Y., Lo W., Margaria J.P., De Santis M.C., Zanini C., Forni M., Novelli F., Pece S., Di Fiore P.P., Porporato P.E., Martini M., Belabed H., Nazare M., Haucke V., Gulluni F, & Hirsch E. (2022). Phosphoinositide Conversion Inactivates R‐RAS and Drives Metastases in Breast Cancer. Advanced Science, 9(9), 2103249.

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Western Blot Analysis of Cell Signaling

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After desired treatments as specified as indicated, cells were washed twice with PBS and lysed in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride). Equal amounts of protein (30 µg) were loaded onto 15% SDS-PAGE gels. Western detection was carried out using a Li-Cor Odyssey image reader (Li-Cor, USA). Anti-Rb, anti-Bad, anti-myc, anti-Ras and anti-β-Actin antibodies were obtained from Cell Signaling Technology (CST, USA), and were used with a dilution of 1∶1000. Anti-Bax, anti-Bak and anti-Bcl-XL antibodies were from Bioss (China), and were diluted by 1∶100. The goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG secondary antibodies were obtained from Li-Cor (USA). The final concentration of the secondary antibodies used was 0.1 µg/ml.

Zhu Z., Wang Y., Liang Z., Wang W., Zhang H., Li B, & Ying G. (2014). Regulation of Cell Transformation by Rb-Controlled Redox Homeostasis. PLoS ONE, 9(7), e102582.

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Western Blot Protein Analysis

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The cells were lysed in RIPA buffer with protease and phosphatase inhibitors (1:100). The BCA kit (#P0010, Beyotime) detected protein samples. Proteins were loaded on 7.5%–12% SDS-PAGE gels and then transferred to PVDF membranes (EMD Millipore, Germany). The membrane was then blocked with 5% skimmed milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight (anti-Ras, #67648, 1:1000, Cell Signaling Technology; anti-ASXL3, #C6072DI210-1, 1:1000, Genscript; anti-P-ERK, #9106, 1:2000, Cell Signaling Technology; anti-FGFR2, #A12436, 1:1000, Abclonal; anti-ERK, #9102, 1:2000, Cell Signaling Technology; anti-GAPDH, #60004-1-lg, 1:10000, ProteinTech). The second antibodies were added and incubated at room temperature for 1 h. Protein bands were detected using the ECL Substrate Kit (Thermo Fisher Scientific, Massachusetts).

Liu Z., Jiang Y., Fang F., Li R., Han J., Yang X., Deng Q., Li L.S., Lei T.Y., Li D.Z, & Liao C. (2023). ASXL3 gene mutations inhibit cell proliferation and promote cell apoptosis in mouse cardiomyocytes by upregulating lncRNA NONMMUT063967.2. Biochemistry and Biophysics Reports, 35, 101505.

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Comprehensive Molecular Signaling Analysis

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All the antibodies used in the present study, including anti-MEK (catalog number, 4694) and anti-ERK (catalog number, 9102) with their Phospho-antibody (catalog number, 3598 and 4370 respectively), anti-Ras (catalog number, 3965), anti-cyclinD1 (catalog number, 2922), anti-cyclinE1 (catalog number, 4129), anti-cleaved caspase-3 (catalog number, 9661) and anti-cleaved pARP1 (catalog number, 5625), anti-β-actin (catalog number, 3700), were purchased from Cell Signaling Technology. Antibody against BTG2 was purchased from Santa Cruz Biotechnologies (catalog number, sc-33775). Dual Luciferase Reporter Gene Assay Kit was purchased fromBeyotime Biotechnology. TUNEL FITC Apoptosis Detection Kit and Cell Cycle Assay Kit were purchased from Vazyme. PCR Reagents and restriction endonucleases were purchased from Takara. PRL-TK vectors and pmirGLO Vector were purchased from Promega. PEGFP-C1 was purchased fromClontech Laboratories Inc and PcDNA 3.1(+) was purchased from Invitrogen. The miRNA mimics and inhibitor were purchased from Biotend Biotechnologies.

Zhou L., Liang X., Zhang L., Yang L., Nagao N., Wu H., Liu C., Lin S., Cai G, & Liu J. (2016). MiR-27a-3p functions as an oncogene in gastric cancer by targeting BTG2. Oncotarget, 7(32), 51943-51954.

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