P44 p42 mapk

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P44/p42 MAPK is a protein that functions as an extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) signaling pathway. It plays a role in the regulation of various cellular processes, including cell proliferation, differentiation, and survival.

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Anti-phospho-p42/p44 MAPK (6 citations) P-p44/p42 MAPK (5 citations) Anti–Phospho-p44/p42 MAPK (anti-pTEpY) (5 citations) Phospho p42/p44 MAPK (5 citations) Anti-p44/p42 MAPK (5 citations) Anti-phospho-p44/p42 MAPK-Thr202/Tyr204 (4 citations) Phospho-P44/P42 MAPK (ERK 1/2) (3 citations) Anti–phospho-p44/p42 MAPK (Thr202/Tyr204) antibody (3 citations) Anti-phospho-p44/p42 MAPK antibody (3 citations) Rabbit anti-phospho-p44/p42 MAPK antibody (2 citations)

14 protocols using p44 p42 mapk

Quantification of Phosphorylated ERK Levels

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Extracts from cortical tissue were prepared using RIPA buffer (Thermo Scientific, Rockford, IL), protease inhibitors (Complete Mini, Roche, Mannheim, Germany) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set III, Calbiochem, San Diego, CA). Samples were homogenized using a Pro Homogenizer (ProScientific Inc., Oxford, CT) and protein concentration was determined using the bicinchoninic acid (BCA) assay (ThermoScientific) with bovine serum albumin as a standard. 30 µg of protein was loaded per lane on a 4–12% gradient acrylamide gel. Proteins were transferred to a nitrocellulose membrane and probed with an anti-phosphorylated ERK (p44/p42 MAPK, #9101, Cell Signaling Technology, Danvers, MA) or an anti-ERK (p44/p42 MAPK, #4695, Cell Signaling Technologies) specific antibody. Western blots were imaged and quantitated using the Licor Odyssey Infrared Imaging System and pERK was normalized to total ERK. Combined data are expressed as the mean ± SEM and Student’s t-test was used to detect significant (p<0.05) differences in the levels of pERK.

Humphreys G.I., Ziegler Y.S, & Nardulli A.M. (2014). 17β-Estradiol Modulates Gene Expression in the Female Mouse Cerebral Cortex. PLoS ONE, 9(11), e111975.

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Western Blot Analysis of Cell Signaling

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Cells were lysed in the M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) containing halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined by the BCA reagents (Thermo). About 10 μg proteins were denatured and applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis products were transferred to a polyvinyl idenefluoride (PVDF) film and PVDF membranes were then incubated at room temperature with specific primary antibody. After a standard washing, membranes were incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The assay developed using a chemiluminescent substrate. The primary antibodies used in this study included antibodies against β-actin, p-p44/p42 MAPK, p44/p42 MAPK, p-p38 MAPK, p38 MAPK, p-JNK, JNK (Cell Signaling Technology), EV71 VP1 (Abnova) and COX-2 (Santa Cruz Biotechnology). The goat anti-rabbit and anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology.

Wang H., Zhang D., Ge M., Li Z., Jiang J, & Li Y. (2015). Formononetin inhibits enterovirus 71 replication by regulating COX- 2/PGE2 expression. Virology Journal, 12, 35.

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Antibody Characterization for Cell Signaling

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Polyclonal anti-TOM1L1 (1: 2,000) antibodies were as described in ref. 23 (link). The monoclonal anti-TOM1L1 antibody was from Covalab (Lyon, France). Antibodies against ERBB2 (1:1,000), p-CortactinY421 (1:500), p-AKTS473 (1:1,000), AKT (1:1,000), P44/P42 MAPK (1:1,000) and p-P44/P42 MAPK (1:1,000) were from Cell Signaling Technology (Danvers, USA). Antibody against Cortactin (1:500) was from Millipore (Billeria, USA) and antibodies against MT1-MMP (1:100 for if or 1:500 for immunoblotting) and TOLLIP (1:200) from Abcam (Cambridge, UK). Anti-tubulin (1:2,000), HA-tag (1:4,000) and p-Tyr 4G10 (1:50) antibodies were from N. Morin, C. Gauthier-Rouvière and P. Mangeat, respectively (CRBM, Montpellier, France). Anti-rabbit IgG-HRP (1:5,000) and anti-mouse IgG-HRP (1:5,000) were from GE Healthcare (Fairfield, USA). Anti-rabbit and anti-mouse IgG coupled to Alexa-Fluor 488, Alexa-Fluor 594 and Alexa-Fluor 405 (1:1,000) were from Life Technologies (Carlsbad, USA). Alexa-Fluor 594-Phalloidin (Life Technologies) was used to visualize F-actin. GFP-Nanotrap technology antibodies were from Chromotek (Planegg-Martinsried, Germany).

Chevalier C., Collin G., Descamps S., Touaitahuata H., Simon V., Reymond N., Fernandez L., Milhiet P.E., Georget V., Urbach S., Lasorsa L., Orsetti B., Boissière-Michot F., Lopez-Crapez E., Theillet C., Roche S, & Benistant C. (2016). TOM1L1 drives membrane delivery of MT1-MMP to promote ERBB2-induced breast cancer cell invasion. Nature Communications, 7, 10765.

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Immunoblotting Protocol for Inflammatory Signaling

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Lysates were prepared in RIPA buffer (Cell Signaling Technologies) with 1mM PMSF per manufacturer's instructions. Proteins were separated on a NuPAGE gel (Invitrogen) and transferred to a PVDF membrane using the XCell II blotting system (Invitrogen). Membranes were blocked with 5% nonfat milk and incubated with primary antibody overnight at 4°C. Primary antibodies were purchased from Cell Signaling Technologies: IRAK1 (#4504), phospho-IkBa (#2859), IkBa (#4814), phospho-p38 MAPK (#4511), p38 MAPK (#8690), phospho-p44/42 MAPK (ERK1/2, #4370), p44/p42 MAPK (ERK1/2, #4695), NFkB2 p100/p52 (#4882), phospho-NFkB p65 (#3033), NFkB p65 (#8242) phospho-TBK1 (#5483), TBK1 (#3504). CXCL1 (PA1-29220, Thermo Fisher Scientific), GAPDH (CB1001), and total actin antibody (MAB1501) were purchased from EMD Millipore. HuR (sc-5261), IRAK1 (sc-5288), phospho-MAPKAPK2 (sc-293139), MAPKAPK2 (sc-393609), and TTP (sc-374305) were purchased from Santa Cruz Biotechnology. TRAF2 (592) was purchased from Medical & Biological Laboratories. Following washing, membranes were incubated with HRP-tagged anti-mouse IgG (1706516, Bio-Rad) or anti-rabbit IgG (NA934, GE Healthcare) and developed using SuperSignal West Pico or Femto substrate (Thermo Fisher Scientific).

Hornick E.E., Banoth B., Miller A.M., Zacharias Z.R., Jain N., Wilson M.E., Gibson-Corley K.N., Legge K.L., Bishop G.A., Sutterwala F.S, & Cassel S.L. (2017). Nlrp12 mediates adverse neutrophil recruitment during influenza virus infection. Journal of immunology (Baltimore, Md. : 1950), 200(3), 1188-1197.

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Western Blot Analysis of Signaling Pathways

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The detailed protocol has been described previously^{31 (link)}. The primary antibodies used for western blot analyses were as follows: EGFR (catalog #4267), phospho- (p-)EGFR (Tyr1068) (#3777), Stat3 (#12640), p-Stat3 (Tyr705) (#9145), Akt (#9272), p-Akt (Ser473) (#4060), p44/p42 MAPK (#9102), p-p44 ⁄ p42 MAPK (#9101), Cyclin B1 (#4138), Cyclin D1 (#2922), Bcl-2 (#2872) (Cell Signaling Technology, Danvers, MA, USA), S100A11 (Medical & Biological Laboratories, Nagoya, Japan, catalog #CY-M1037), and β-actin (used as a loading control) (Merck Millipore, Billerica, MA, USA, catalog # MAB1501). The following secondary antibodies were used: goat anti-rabbit (catalog #sc-2030) or anti-mouse (catalog #sc-2031) immunoglobulin G (IgG)-conjugated horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK, Code No. RPN2235) and LAS-3000 imager (Fujifilm, Tokyo, Japan).

Sato H., Sakaguchi M., Yamamoto H., Tomida S., Aoe K., Shien K., Yoshioka T., Namba K., Torigoe H., Soh J., Tsukuda K., Tao H., Okabe K., Miyoshi S., Pass H.I, & Toyooka S. (2018). Therapeutic potential of targeting S100A11 in malignant pleural mesothelioma. Oncogenesis, 7(1), 11.

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Protein Expression Profiling in Cell Lysates

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Cell lysates were prepared using a buffer containing 10 mmol/L Tris-HCl at pH 7.4 and 1% sodium dodecyl sulfate (SDS). Protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed on the cell lysates, transferred to polyvinylidene fluoride membranes, and immunostained overnight using antibodies as described below. The following antibodies were purchased from Cell Signaling Technology: p21 (dilution at 1:1,000), p27 (1:1,000), cyclinB1 (1:1,000), aurora kinase A (AURKA; 1:1,000), polo-like kinase 1 (PLK1; 1:1,000), cyclin-dependent kinase 1 (CDK1; 1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), acetyl-histone H3 (1:1,000), histone H3 (1:1,000), p44/p42 MAPK (ERK1/2; 1:1,000), p-p44/p42 MAPK (ERK1/2; 1:1,000), p-AKT1 (1:1,000), and AKT1 (1:1,000). Aurora kinase B (AURKB; 1:1,000) and HDAC2 (1:1,000) antibodies were purchased from Abcam. Survivin (1:1,000) was obtained from Novus Biologicals, N-cadherin (1:1,000) from EMD Millipore, HDAC1 (1:1,000) from Thermo Fisher Scientific, and HDAC10 from BioVision. The following antibodies were purchased from Santa Cruz Biotechnology: p27(1:1,000), HDAC4(1:1,000), HDAC6 (1:1,000), and GAPDH (1:5,000). Band densitometry analysis was performed using ImageJ software (NCI).

Kotian S., Zhang L., Boufraqech M., Gaskins K., Gara S.K., Quezado M., Nilubol N, & Kebebew E. (2017). Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits Thyroid Cancer Growth and Metastases. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5044-5054.

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Protein Expression Analysis by Western Blot

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Tissue lysate containing 30 μg protein was separated by SDS–PAGE and transferred to Immun-Blott PVDF membrane (GE Healthcare). The membrane was blocked in 5% milk for 1 h and then incubated with primary antibody (1:1000 dilution) of p53, phospho p53, Bax, Bcl-2, caspase 3, cleaved caspase 3, PARP, p44/p42 MAPK, phospho p44/p42 MAPK, c-Myc, phospho (serine 62) c-Myc, p21 ^waf1/cip1, cyclin D1, phospho Rb (807/811) (Cell Signalling Technologies), anti-GTPase Hras (Abcam), respectively, as needed. GAPDH (Biobharati, India) was used as a loading control. After washing three times with TBS-T (20 mM Tris, 500 mM NaCl, 0.1% Tween-20, pH 7.5), the membrane was incubated with HRP-conjugated secondary antibody (1:3000 dilution) for 1 h at room temperature. The membrane was further washed with TBS-T and developed with Lumiglo reagent (Cell Signaling Technologies).

Ganguly S., Chandra A., Chattopadhyay D.J, & Chatterjee I.B. (2017). p-Benzoquinone initiates non-invasive urothelial cancer through aberrant tyrosine phosphorylation of EGFR, MAP kinase activation and cell cycle deregulation: Prevention by vitamin C. Toxicology Reports, 4, 296-305.

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Western Blot Analysis of Inflammatory Signaling

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Western blot analysis for TNF (1:2,000, Abcam) was performed using 20 μg protein extract separated on 4–12% SDS-PAGE gels (Nupage, Invitrogen) essentially as previously described3 (link)4 (link). As a positive control, 0.5 ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included. Western blotting analysis for SAPK/JNK (Cell Signaling, 1:1,000), phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (Cell Signaling, 1:1,000), p44/p42 MAPK (Cell Signaling, 1:1,000), p-p44/p-p42 MAPK (ERK1/2)(Cell Signaling, 1:1,000), p-38 (Cell Signaling, 1:1,000), p-p38 MAPK (Tyr180/Tyr182)(Cell Signaling, 1:1,000), and Iba1 (Wako, 1:500) was performed by resolving equal amounts of protein lysates by SDS-PAGE on Nupage Bis 4–12% gels, using MOPS SDS (Invitrogen) containing 0.25% antioxidant (Invitrogen) essentially as previously described6 (link)59 (link). TFIIB (1:1,000, Cell Signaling), GAPDH (1:2,500, Abcam) and α-actin (1:8,000, Millipore) were used as loading controls. SeeBlue Plus2 prestained standard (Invitrogen) was used as a molecular weight marker. Bands were quantified with Quantity One software (Biorad). Analysis was performed on 2–4 independent gels with n = 2–3/group and data were normalized to TFIIB, GAPDH or α-actin and represented as percentages relative to naïve TNF^fl/fl mice or as ratios (for MAPK).

Clausen B.H., Degn M., Sivasaravanaparan M., Fogtmann T., Andersen M.G., Trojanowsky M.D., Gao H., Hvidsten S., Baun C., Deierborg T., Finsen B., Kristensen B.W., Bak S.T., Meyer M., Lee J., Nedospasov S.A., Brambilla R, & Lambertsen K.L. (2016). Conditional ablation of myeloid TNF increases lesion volume after experimental stroke in mice, possibly via altered ERK1/2 signaling. Scientific Reports, 6, 29291.

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Western Blot Analysis of Signaling Proteins

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The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma‐Aldrich, St. Louis, MO, USA), and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was carried out by conventional methods using the following primary antibodies: anti‐EGFR, phospho‐ (p‐)EGFR (Tyr1068), HER2, p‐HER2 (Tyr1221/1222), Akt, p‐Akt (Ser473), p44/p42 MAPK, p‐p44/p42 MAPK, cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA, USA), and β‐actin (used as loading control) (Merck Millipore, Billerica, MA, USA). The secondary antibody was HRP‐conjugated anti‐mouse or anti‐rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS‐3000 (Fujifilm, Tokyo, Japan).

Suzawa K., Toyooka S., Sakaguchi M., Morita M., Yamamoto H., Tomida S., Ohtsuka T., Watanabe M., Hashida S., Maki Y., Soh J., Asano H., Tsukuda K, & Miyoshi S. (2015). Antitumor effect of afatinib, as a human epidermal growth factor receptor 2‐targeted therapy, in lung cancers harboring HER2 oncogene alterations. Cancer Science, 107(1), 45-52.

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Signaling Pathway Reagent Acquisition and Use

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TGF-β was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Onalespib was purchased from Selleckchem (Houston, TX, USA). Geldanamycin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 17-DMAG and SP600125 were purchased from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). SB431542 was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). HSP27 antibodies, phosphorylated HSP27 antibodies, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Phospho-specific Smad2, Smad2, phospho-specific p44/p42 MAPK, p44/p42 MAPK, phospho-specific p38 MAPK antibodies, p38 MAPK antibodies, phospho-specific SAPK/JNK antibodies, and SAPK/JNK antibodies, were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfont, UK). Other materials and chemicals were obtained from commercial sources. Onalespib, Geldanamycin, 17-DMAG, SP600125, and SB431542 were dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the assay for Western blot analysis [21 (link)–23 (link)].

Kuroyanagi G., Tokuda H., Fujita K., Kawabata T., Sakai G., Kim W., Hioki T., Tachi J., Matsushima-Nishiwaki R., Otsuka T., Iida H, & Kozawa O. (2022). Upregulation of TGF-β-induced HSP27 by HSP90 inhibitors in osteoblasts. BMC Musculoskeletal Disorders, 23, 495.

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