Anti phospho p44 42 mapk erk1 2

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-Phospho-p44/42 MAPK (Erk1/2) is a primary antibody that detects endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when phosphorylated at Thr202/Tyr204 and Thr185/Tyr187, respectively.

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39 protocols using anti phospho p44 42 mapk erk1 2

Characterization of OTUB1 Regulation

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Full‐length OTUB1 expression constructs were purchased from ORIGENE. Point mutations to generate OTUB1 C91S were obtained by QuickChange site‐directed mutagenesis PCR (Stratagene, La Jolla). Lentiviral pLA‐CMV‐N‐Flag or pLA‐CMV‐N‐HA vectors were used to generate Flag/HA‐tagged constructs. The pMT107–6×His–ubiquitin plasmid was a generous gift from Dr. Bohmann (University of Rochester, USA). pLKO.1‐puro shGFP and pLKO.1‐puro vectors containing shRNAs targeting OTUB1 (pLKO.1‐shOTUB1_1 (TRCN0000004211), pLKO.1‐shOTUB1_2 (TRCN0000004213), pLKO.1‐shOTUB1_3 (TRCN0000004215)) were purchased from Sigma‐Aldrich.
The antibodies used: mouse monoclonal anti‐FLAG (Sigma‐Aldrich, M2), anti‐RAS (Millipore, Clone 10), anti‐vinculin (Sigma‐Aldrich, clone hVIN‐1), anti‐GAPDH (Sigma‐Aldrich, GAPDH‐71.1), anti‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, 3A7, #9107), anti‐AKT (Cell Signaling, 40D4); rat monoclonal anti‐HA (Roche, 3F10); rabbit monoclonal anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (IHC) (Cell Signaling, D13.14.4E, #4370), anti‐Ki67 (Thermo Scientific #RM‐9106‐S, clone SP6); rabbit polyclonal anti‐DYKDDDDK (Cell Signaling), anti‐OTUB1 (Bethyl Laboratories), anti‐OTUB1 (IHC) (Sigma‐Aldrich, HPA039176), anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, #9101), anti‐pAKT (Cell Signaling, D9E), anti‐RABEX5 (Sigma‐Aldrich).

Baietti M.F., Simicek M., Abbasi Asbagh L., Radaelli E., Lievens S., Crowther J., Steklov M., Aushev V.N., Martínez García D., Tavernier J, & Sablina A.A. (2016). OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination. EMBO Molecular Medicine, 8(3), 288-303.

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Quantitative Protein Analysis in Cells

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Western blotting was performed as previously described [50 (link)]. The protein concentration was measured using the DC protein assay (Bio-Rad, Hercules, CA, USA). The primary antibodies included anti-Ucp1 (Sigma-Aldrich, St. Louis, MO, USA), anti-COXIV, anti-phospho-p44/42 MAPK (ERK1/2), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-Pparγ, and anti-β-actin (all purchased from Cell Signaling Technology, Danvers, MA, USA). The secondary antibody staining was visualized using a chemiluminescent horseradish peroxidase substrate (Millipore, Burlington, MA USA).

Yuliana A., Daijo A., Jheng H.F., Kwon J., Nomura W., Takahashi H., Ara T., Kawada T, & Goto T. (2019). Endoplasmic Reticulum Stress Impaired Uncoupling Protein 1 Expression via the Suppression of Peroxisome Proliferator-Activated Receptor γ Binding Activity in Mice Beige Adipocytes. International Journal of Molecular Sciences, 20(2), 274.

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Comprehensive Protein Expression Analysis

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Whole cell extract was prepared using RIPA buffer (Thermo Scientific) with a Halt^TM Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) [15 (link)]. Laemmli’s sample buffer was added to the cell lysates and boiled at 95 °C for 5 min. The cell lysates were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies against anti-EGF Receptor, anti-phospho-EGF Receptor (Tyr1068), anti-HER2/ErbB2, anti-HER3/ErbB3, anti-HER4/ErbB4, anti-phospho-MEK1/2 (Ser217/221), anti-MEK1/2, anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-p44/42 MAPK (Erk1/2), anti-Akt, anti-phospho-Akt (Ser473), anti-PARP, anti-Caspase-3 and anti-β-actin (Cell Signaling).

Liu C.Y., Chu P.Y., Huang C.T., Chen J.L., Yang H.P., Wang W.L., Lau K.Y., Lee C.H., Lan T.Y., Huang T.T., Lin P.H., Dai M.S, & Tseng L.M. (2019). Varlitinib Downregulates HER/ERK Signaling and Induces Apoptosis in Triple Negative Breast Cancer Cells. Cancers, 11(1), 105.

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Molecular Mechanisms of 4-Octyl Itaconate

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4-Octyl itaconate was initially supplied by Professor Richard Hartley and results were later confirmed with commercially available 4-OI (Sigma Aldrich). Pam3CSK4, dimethyl fumarate, diethyl maleate, indomethacin and NS-398 (Sigma Aldrich) were also used. Antibodies used were anti-β-actin (Sigma Aldrich), anti-COX2 (Abcam), anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-NRF2, anti-KEAP1, anti-ATF4, anti-phospho-NF-kB p65 (Ser536), anti-NF-kB p65, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK, anti-phospho p44/42 MAPK (Erk1/2) (Thr202/Tyr 204) and anti-p44/42 MAPK (Erk1/2) (Cell Signaling). Anti-mouse IgG and anti-rabbit IgG secondary horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch) were also used. A PGE₂ ELISA kit was used (Enzo Life Sciences). The Silencer Select siRNAs against NRF2 (s70522), ATF4 (s62689) and Anxa1 (s69299), as well as the Silencer Select negative control, were used (ThermoFisher Scientific).

Diskin C., Zotta A., Corcoran S.E., Tyrrell V.J., Zaslona Z., O’Donnell V.B, & O’Neill L.A. (2021). 4-Octyl-itaconate and dimethyl fumarate inhibit COX2 expression and prostaglandin production in macrophages. Journal of immunology (Baltimore, Md. : 1950), 207(10), 2561-2569.

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Western Blot Analysis of Protein Signaling

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Cells were lysed in ice‐cold RIPA buffer (Beyotime Biotechnology) and supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology). Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology). Total proteins were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After blocking, the membranes were incubated with a primary antibody and detected with a horseradish peroxidase‐conjugated secondary antibody. Proteins were visualized using ECL Western blotting detection kit (Advansta). Immunoreactive bands were quantified using Image Quant TL software. The following antibodies were used: anti‐PLPPR4 (Jackson, sc‐377263, 1:1000), anti‐phospho‐p44/42 MAPK (ERK1/2) (Cell Signaling Technology, 4370 s, 1:1000), anti‐p44/42 MAPK (ERK1/2) (Cell Signaling Technology, 4659, 1:1000), anti‐phospho‐mTOR (Cell Signaling Technology, 2971S, 1:1000), anti‐mTOR (Cell Signaling Technology, 2983S, 1:1000) and anti‐GAPDH (Cell Signaling Technology, 5174S, 1:1000). All antibodies were diluted in PBS containing 5% skimmed milk and 0.1% Tween‐20.

Li H., Zhang Q., Wan R., Zhou L., Xu X., Xu C., Yu Y., Xu Y., Xiang Y, & Tang S. (2023). PLPPR4 haploinsufficiency causes neurodevelopmental disorders by disrupting synaptic plasticity via mTOR signalling. Journal of Cellular and Molecular Medicine, 27(21), 3286-3295.

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Immunohistochemical Analysis of Cell Cycle and Angiogenesis Markers

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FFPE blocks were cut into 5‐μm‐thick sections for immunohistochemistry (IHC) studies. The cell cycle in normal tissue was tested with anti‐p21 (WAF1) from Merck (Darmstadt, Germany; Ref MABE325), anti‐p27 (57/Kip1) from BD Biosciences (Franklin Lakes, NJ; Ref 610242), and anti–phosphohistone γH2AX (Ser139) from Millipore (Burlington, MA; Ref 05‐636) antibodies. Activation of the VEGF‐angiogenesis pathway was assessed with 2 different antibodies against phosphorylated (activated) proteins: anti–phospho‐p44/42‐MAPK (ERK1/2) and anti–phospho‐S6 ribosomal protein (Ser235/236) from Cell Signaling Technology (Danvers, MA; Refs 9101 and 2211, respectively). Both the absence of staining and excess nonspecific staining were considered as negative staining. Staining was considered separately in normal (N) and tumor (T) tissues. IHC controls were performed by using normal tissue from biopsies of healthy individuals.

Calvete O., Garcia‐Pavia P., Domínguez F., Mosteiro L., Pérez‐Cabornero L., Cantalapiedra D., Zorio E., Ramón y Cajal T., Crespo‐Leiro M.G., Teulé Á., Lázaro C., Morente M.M., Urioste M, & Benitez J. (2019). POT1 and Damage Response Malfunction Trigger Acquisition of Somatic Activating Mutations in the VEGF Pathway in Cardiac Angiosarcomas. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(18), e012875.

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Western Blot Antibody Procurement

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Anti-iNOS (PA1-036) was purchased from Invitrogen (Carlsbad, CA, USA). Anti-phospho-p44/42 MAPK (Erk1/2) (#9101), anti-p44/42 MAPK (Erk1/2) (#4695), anti-phospho-SAPK/JNK (#9251), anti-SAPK/JNK (#9252), anti-phospho-p38 MAPK (#9211), anti-p38 MAPK (#9212), anti-phospho-NF-κB p65 (#3033), anti-phospho-IκBα (#2859), anti-IκBα (#4814), anti-phospho-JAK1 (#3331), anti-JAK1 (#3332), anti-phospho-STAT1 (#9167), anti-STAT1 (#9172), anti-phospho-STAT3 (#9145), and anti-STAT3 (#30835) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NFκB p65 (sc-8008) and anti-β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Carlsbad, CA, USA). HRP-conjugated anti-mouse IgG (A21010) and HRP-conjugated anti-rabbit IgG (A21020) were purchased from Abbkine (Wuhan, China). The information on antibodies is summarized in Supplementary Table S2.

Kim S.H., Jang Y.A, & Kwon Y.J. (2024). Anti-Inflammatory Effect of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Essential Oil. Molecules, 29(5), 1117.

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Western Blot Reagents and Antibodies

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FXa was obtained from Hematologic Technologies. Anti-phospho-p44/42 MAPK (Erk1/2) and anti-ERK1/2 Abs were obtained from Cell Signaling Technology. GAPDH Ab was obtained from Santa Cruz Biotechnology. Secondary Abs, HRP Goat anti-Rabbit IgG and HRP Goat anti-Mouse IgG, were obtained from Invitrogen, USA and Seracare, USA, respectively.

Shrivastava G., Valenzuela-Leon P.C., Chagas A.C., Kern O., Botello K., Zhang Y., Martin-Martin I., Oliveira M.B., Tirloni L, & Calvo E. (2022). Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus, Displays Anti–FXa-PAR Signaling In Vitro and In Vivo. ImmunoHorizons, 6(6), 373-383.

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Western Blot Analysis of Phospho-Signaling

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Cell pellets were lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, Halt protease and phosphatase inhibitor cocktail [1:100]). Protein extracts (50 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes using the trans-blot turbo transfer system (Bio-Rad). Membranes were blocked with 5% dry milk in TBST, followed by incubation with primary and fluorescence-labeled secondary antibodies. Fluorescence signals were imaged using the Odyssey CLx western blot detection system (LI-COR). The following antibodies were used: anti-phospho-MEK1/2 (Cell Signaling Technology 9121), anti-MEK1/2 (Cell Signaling Technology 4694), anti-phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; Cell Signaling Technology 4376), anti-p44/42 MAPK (ERK1/2; Cell Signaling Technology 4696), anti-HSP90 (Santa Cruz sc-7947), goat anti-rabbit IgG DyLight 680 Conjugate (Cell Signaling Technology 5366S), and anti-mouse IgG DyLight 800 4x PEG Conjugate (Cell Signaling Technology 5257S).

Uhrig S., Ellermann J., Walther T., Burkhardt P., Fröhlich M., Hutter B., Toprak U.H., Neumann O., Stenzinger A., Scholl C., Fröhling S, & Brors B. (2021). Accurate and efficient detection of gene fusions from RNA sequencing data. Genome Research, 31(3), 448-460.

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Immunoblotting for Protein Fractionation

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Immunoblotting was performed as previously described [18]. Cell lysates were extracted with a lysis buffer (1% Nonidet P‐40, 150 mm NaCl, and 20 mm Tris/HCl). To obtain the cytoplasmic and nuclear fractions, the NE‐PER™ Nuclear and Cytoplasmic Extraction Reagents (#787833; Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The protein concentrations were measured with the BCA Protein Assay (Pierce, Thermo Fisher Scientific). Proteins were applied to SDS/polyacrylamide gel electrophoresis and transferred to a membrane. Anti‐NTSR1 (PA3‐214; Thermo Fisher Scientific), anti‐phospho‐p44/42 MAPK (Erk1/2) (#9101; Cell Signaling Technology, Danvers, MA, USA), anti‐ERK (#9102; Cell Signaling Technology), anti‐phospho‐p38 MAP kinase (#9211; Cell Signaling Technology), anti‐p38 (#9228; Cell Signaling Technology), anti‐phospho‐SAPK/JNK (#9251; Cell Signaling Technology), anti‐p65 (#8242; Cell Signaling Technology), anti‐HDAC1 (clone 2E10; Sigma‐Aldrich, Merck Millipore), and anti‐α‐tubulin (T9026; Sigma‐Aldrich, Merck Millipore) antibodies were used. The bands were quantified with Multi Gauge software (FUJIFILM) and analyzed three times with subtraction of the background signal intensity.

Takahashi K., Ehata S., Miyauchi K., Morishita Y., Miyazawa K, & Miyazono K. (2020). Neurotensin receptor 1 signaling promotes pancreatic cancer progression. Molecular Oncology, 15(1), 151-166.

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