Phospho erk1 2

Manufactured by Merck Group

Sourced in United States

Phospho-Erk1/2 is a laboratory product used for the detection and quantification of phosphorylated extracellular signal-regulated kinase 1 and 2 (Erk1/2) proteins. It is designed to facilitate research and analysis related to cellular signaling pathways.

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Lab products found in correlation

Phospho akt, by Merck Group (3 mentions) Anti il4 blocking antibody clone 11b11, by Thermo Fisher Scientific (2 mentions) Anti h3k27ac ab4729 antibody, by Abcam (2 mentions) Phospo stat1, by Merck Group (2 mentions) Stat6, by Merck Group (2 mentions) Phospho stat6, by Merck Group (2 mentions) Phospho stat1 alexafluor 647, by BD (2 mentions) Phospho stat6 pe, by BD (2 mentions) Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions) Recombinant mouse ifn γ and il 4, by R&D Systems (2 mentions) Stat1, by Merck Group (2 mentions) Anti actin a4700, by Merck Group (2 mentions) β actin, by Merck Group (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Protease inhibitor mixture, by Merck Group (1 mentions) Anti mouse igg, by Bio-Rad (1 mentions) Erk1 2, by Merck Group (1 mentions) Phospho egfr, by Merck Group (1 mentions) Anti ddx3x, by Cell Signaling Technology (1 mentions) Anti rabbit igg conjugated to horse radish peroxidase, by Abcam (1 mentions)

7 protocols using phospho erk1 2

Signaling Mechanisms in Macrophage Activation

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Recombinant mouse IFN-γ and IL-4 were from R&D Systems. Unless otherwise stated, cytokines were used at a concentration of 100ng/ml and 10ng/ml, respectively. Anti-IL4 blocking antibody (clone 11B11) was from eBioscience. CD11B (clone M1/70), F4/80 (BM8), NOS2 (CXNFT) antibodies for flow cytometry were from eBioscience. Antibodies used in ChIP experiments against Stat1 (sc-592), Stat6 (sc-981), MYC (sc-764), JUNB (sc-46x), C/EBPβ (sc-150X) were all from Santa Cruz. The anti-H3K27Ac (ab4729) antibody was from Abcam. The anti-Pu.1 rabbit polyclonal antibody was generated in-house against the N-terminus of Pu.1 (aa. 1-100; NP_035485.1) 32 (link). Antibodies used in western blot experiments: Stat1 (#9172), phospo-Stat1 (Tyr701, #9171), Stat6 (#9362), phospho-Stat6 (Tyr641, #9361), Erk1/2 (#9102), phospho-Erk1/2 (Thr202/Tyr204, #9101), phospho-Akt (Ser473, #9271); anti-actin (A4700) from Sigma-Aldrich. The following antibodies were used for flow cytometry: CD11b (clone M1/70), F4/80 (BM8), Nos2 (CXNFT) from eBioscience; phospho-Stat1-AlexaFluor 647 (Cat. 612597) and phospho-Stat6-PE (Cat. 558252) from BD Biosciences. The following antibodies were used for immunofluorescence: Alexafluor 488 goat anti-rabbit secondary antibody (Cat. R37116) and DAPI (Cat. 62247) from Thermo Scientific; phospho-Stat1-AlexaFluor 647 (Cat. 612597) and phospho-Stat6-PE (Cat. 558252) from BD Biosciences.

Piccolo V., Curina A., Genua M., Ghisletti S., Simonatto M., Sabò A., Amati B., Ostuni R, & Natoli G. (2017). Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross talks. Nature immunology, 18(5), 530-540.

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Immunoblotting of EGFR and Akt Signaling

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Cells were harvested and lysed in Nonidet P-40 buffer containing a protease-inhibitor mixture (Sigma). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary antibodies consisted of anti-mouse IgG (BioRad) and anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA). Immunoreactive protein bands were visualized using an ECL kit (Pierce). At least three independent experiments were performed for all analyses.

Nozaki K., Kagamu H., Shoji S., Igarashi N., Ohtsubo A., Okajima M., Miura S., Watanabe S., Yoshizawa H, & Narita I. (2014). DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations. PLoS ONE, 9(10), e111019.

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Protein Kinase Inhibitors and ELISA

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PD98059 (PD), SB203580 (SB) and SP6000125 (SP) were purchased from Calbiochem (San Diego, CA, USA). All enzyme-linked immunosorbent assay (ELISA) kits for phospho-p38 MAPK (Product No: CS0020), total p38 MAPK (Product No: PM0100), Phospho-JNK1&2 (Product No: CS0130), JNK 1&2 (Product No: CS01000), Phospho-ERK1/2 (Product No: CS 7177), total ERK1/2 (Product No: CS 7050), and P-selectin ELISA kit (Catalog Number RAB0426.) were from Sigma Aldrich.

Rashtchizadeh N., Karimi P., Dehgan P, & Salimi Movahed M. (2015). Effects of Selenium in the MAPK Signaling Cascade. Journal of Cardiovascular and Thoracic Research, 7(3), 107-112.

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Signaling Mechanisms in Macrophage Activation

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Inhibitors and Antibodies for Cellular Signaling

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PLD1i (VU0359595) and PLD2i (VU0285655-1) were from Avanti Polar Lipids (Alabaster, AL, USA). Antibodies for phosphorylated and/or total AKT (1 : 1000), p70S6K (1 : 1000), 4E-BP1 (1 : 1000), p38 (1 : 1000), ATG5 (1 : 1000), and LC3 (1 : 1000) were from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal p62 antibody was from BD Biosciences (San Jose, CA, USA). FIPI, antibodies for α-tubulin (1 : 5000), phospho-ERK1/2 (1 : 40 000), and total ERK1/2 (1 : 40 000) were from Sigma-Aldrich (St. Louis, MO, USA). LAMP1 was from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Rabbit monoclonal PLD1 antibody (1 : 1000) was from Abcam (Cambridge, MA, USA). Rabbit polyclonal PLD2 antibody (1 : 500) has been described before.^{40 (link), 41 (link)} Goat anti-mouse and anti-rabbit IgGs conjugated with Alexa Fluor 488 or 594 were from Life Technologies (Grand Island, NY, USA). Goat anti-mouse and anti-rabbit IgGs conjugated with IRDye 680CW or IRDye 800CW (1 : 5000) were from Rockland Immunochemicals (Gilbertsville, PA, USA).

Cai M., He J., Xiong J., Tay L.W., Wang Z., Rog C., Wang J., Xie Y., Wang G., Banno Y., Li F., Zhu M, & Du G. (2016). Phospholipase D1-regulated autophagy supplies free fatty acids to counter nutrient stress in cancer cells. Cell Death & Disease, 7(11), e2448-.

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Protein Extraction and Western Blot Analysis

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The proteins of cells and EVs were extracted with the RIPA as described in the previous study [24 (link)]. The total protein levels of the lysates were measured by BCA Protein Assay Kit (iNtRON Biotechnology, Seongnam-si, South Korea, Cat No. 21071). The following primary antibodies were used at 1:1000 dilution: Alix (Cell Signaling Technology, Danvers, MA, USA, Cat No. 2171), CD63 (Abcam, Cambridge, UK, Cat No. ab59479), CD9 (Abcam, Cat No. ab92726), CD81 (Abcam, Cat No. ab79559), VEGFA (Abcam, Cat No. ab214424), VEGFR1 (Abcam, Cat No. ab32152), VEGFR2 (Abcam, Cat No. ab134191), Erk1/2 (Cell Signaling Technology, Cat No. 4695) and β-actin (Invitrogen; Thermo Fisher Scientific, Cat No. MA5-15739-HRP). The primary antibody phospho-Erk1/2 (Sigma-Aldrich, Cat No. M9692) was used at 1:3000 dilution. The secondary antibodies were used at 1:3000 dilution. The intensity of the bands was analyzed by the ImageJ “gel analysis” function. Briefly, multiple bands were selected by identical squares, and the pixel intensity distribution along the y-axis was plotted for each square. Area under the distribution curve was analyzed and normalized by rows so that the sum in each row was consistently 10.

Zhang P., Lim S.B., Jiang K., Chew T.W., Low B.C, & Lim C.T. (2021). Distinct mRNAs in Cancer Extracellular Vesicles Activate Angiogenesis and Alter Transcriptome of Vascular Endothelial Cells. Cancers, 13(9), 2009.

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Immunoblotting and Immunohistochemistry of Cellular Proteins

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The level of protein expression in cultured cells was determined by immunoblotting as previously described [17 (link),18 (link)] using primary antibodies against MMP-9 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), p53, phospho-ERK1/2, total ERK, and β-actin (all Sigma-Aldrich, St. Louis, MO, USA). Protein expression in xenografted tumors was analyzed by immunohistochemistry. Tumor tissues were fixed immediately after harvesting in 10% phosphate-buffered formalin, embedded in paraffin, and tissue sections were stained with the antibodies indicated above.

Liu C., Dai L., Liu Y., Rong L., Dou D., Sun Y, & Ma L. (2016). Antiproliferative Activity of Triterpene Glycoside Nutrient from Monk Fruit in Colorectal Cancer and Throat Cancer. Nutrients, 8(6), 360.

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