Phospho p44 42 mapk

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Phospho-p44/42 MAPK is a lab equipment product that detects the phosphorylation of p44/42 MAPK (Erk1/2) proteins. It is used to measure the activation of the MAPK signaling pathway.

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Close spelling variants of this product

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (97 citations) P44/42 MAPK (90 citations) Anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (49 citations) Anti-Phospho-p44/42 MAPK (Erk1/2) (39 citations) Rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (8 citations) Rabbit anti-phospho p44/42 MAPK (p- Erk1/2), (6 citations) Anti-phospho-p44/42 MAPK (pERK1/2), (6 citations) Anti-phospho-p44/42 MAPK (Erk1/2) antibody (5 citations) Rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr204) (5 citations) Phospho-p44/42 MAPK (pERK) (3 citations)

93 protocols using phospho p44 42 mapk

Immunoblotting Protein Expression Analysis

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Tissues were lysed in RIPA buffer, and concentration of protein lysates were measured using BioRad Protein Assay. Equal concentrations of protein were loaded and electrophoresed on 4–15% TGX Criterion gels (BioRad), transferred to PVDF membranes, and immunoblotted. Antibodies used were PU.1 (2266S, rabbit polyclonal, Cell Signaling), pSer 473 AKT (rabbit monoclonal, Cell Signaling), panAKT (mouse monoclonal, Cell Signaling), pSer 273 PPARg (bs-4888R, rabbit polyclonal, Bioss), PPARg (sc-7196, rabbit polyclonal, Santa Cruz), HSP 90α/β (sc-7947, rabbit polyclonal, Santa Cruz), Phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)(9101, Cell signaling), p-Cdk5(ser159), sc-12919, Santa Cruz), followed by appropriate HRP-linked secondary antibody. SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific) was used to develop blots. Band intensity was measured using NIH ImageJ software.

Lackey D.E., Reis F.C., Isaac R., Zapata R.C., El Ouarrat D., Lee Y.S., Bandyopadhyay G., Ofrecio J.M., Oh D.Y, & Osborn O. (2019). Adipocyte PU.1 knockout promotes insulin sensitivity in HFD-fed obese mice. Scientific Reports, 9, 14779.

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Western Blot Analysis of Signaling Pathways

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Protein lysates from cells were prepared and quantified by Bio-Rad protein assay (Bio-Rad). Proteins (30 μg) were loaded to each lane and separated by electrophoresis on NuPAGE 4 to 12% gels (Thermo Fisher Scientific), followed by transferring to 0.2μm nitrocellulose membrane. Membranes were incubated with 5% nonfat dry milk for 1 hour, washed three times for 5 minutes, and incubated with primary antibodies against phospho-JAK1(Tyr1034/1035) (D7N4Z, 1:1000), JAK1 (D1T6W, 1:1000), phospho-STAT5 (Tyr694) (D47E7, 1:1000), STAT5 (D3N2B, 1:1000), phospho-AKT (Ser473) (D9E, 1:1000), AKT (pan) (C67E7, 1:1000), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E, 1:1000), p44/42 MAPK (Erk1/2) (137F5, 1:1000), β-Actin (8H10D10, 1:1000), (Cell Signaling) overnight at 4 °C. Membranes were washed three times for 5 minutes at room temperature and incubated with horseradish-linked secondary antibodies: anti-mouse IgG, HRP-linked antibody (#7076, 1:3000, Cell Signaling Technology), anti-rabbit IgG, HRP-linked antibody (#7074, 1:3000, Cell Signaling Technology) for 1 hour at room temperature followed by another wash. Bands were visualized using an ECL detection system (Millipore).

Zhang Q., Hresko M.E., Picton L.K., Su L., Hollander M.J., Nunez-Cruz S., Zhang Z., Assenmacher C.A., Sockolosky J.T., Garcia K.C, & Milone M.C. (2021). A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia. Science translational medicine, 13(625), eabg6986.

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Western Blot Analysis of EMT Markers

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Equal quantities of cell extracts were separated on a 10% SDS-PAGE gel, transferred, and analyzed by the Western lightning plus-ECL detection system (Perkin Elmer, Inc., Waltham, MA, USA). Briefly, antibodies against NDRG1, NDRG2, NDRG3 (Invitrogen), Snail (Abcam, Cambridge, UK), cyclin E, E-cadherin, STAT3, N-cadherin, Vimentin (Abgent, San Diego, CA, USA), Slug, p44/42 MAPK, Phospho-p44/42 MAPK, SAPK/JNK, Phospho-SAPK/JNK, p38 MAPK, Phospho-p38 MAPK (Cell Signaling, Danvers, MA, USA), Phospho-STAT3 and β-actin (Millipore) were used and the protein bands were detected and quantified with the ChemiGenius Image Capture System (Syngene, Cambridge, UK) and the GeneTools Program of ChemiGenius (Syngene).

Chiang K.C., Yang S.W., Chang K.P., Feng T.H., Chang K.S., Tsui K.H., Shin Y.S., Chen C.C., Chao M, & Juang H.H. (2018). Caffeic Acid Phenethyl Ester Induces N-myc Downstream Regulated Gene 1 to Inhibit Cell Proliferation and Invasion of Human Nasopharyngeal Cancer Cells. International Journal of Molecular Sciences, 19(5), 1397.

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Detecting IGF System and Differentiation Markers

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In this study, the following antibodies were used to detect the IGF system: phospho-p44/42 MAPK (#4377), p44/42 MAPK (#9102), phospho-AKT (Ser473, #4051), and AKT (#9272) (Cell Signaling Technologies, Burlington, ON) and IGF-1Rα (N-20, sc-712) and IR-α (N-20, sc-710) (Santa Cruz Biotech., Santa Cruz, CA). For multipotency markers, we used OCT3/4 antibody (N-19, sc-8628) (Santa Cruz Biotech., Santa Cruz, CA) and SOX-2 (2683-1) (Epitomics, Burlington, ON). For the osteogenic differentiation markers, we used RUNX2 (#8486) (Cell Signaling Technologies, Burlington, ON), phospho-RUNX2 (PA5-12988) (Thermo Fisher Scientific, Burlington, ON), and OPN (K-20, sc-1059) (Santa Cruz Biotech., Santa Cruz, CA). For the loading control, we used pan-Actin Ab-5 (#MS-1295) (Thermo Fisher Scientific, Fremont, CA). The secondary antibodies used for immunoblotting were goat anti-rabbit (#170-6515), anti-mouse (#170-6516) HRP-conjugated antibodies (Bio-Rad Laboratories, Hercules, CA), or donkey anti-goat antibody (sc-2020) (Santa Cruz Biotech., Santa Cruz, CA).

Youssef A, & Han V.K. (2017). Regulation of Osteogenic Differentiation of Placental-Derived Mesenchymal Stem Cells by Insulin-Like Growth Factors and Low Oxygen Tension. Stem Cells International, 2017, 4576327.

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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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Inhibition of Inflammatory Pathways

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LPS was purchased from Sigma (Escherichia coli 055:B5, USA). IL-1β and TNF-α were from Peprotech (USA). Evans blue, the NF-κB inhibitor PDTC, the p38 inhibitor SB203580, the ERK1/2 inhibitor U0126, and the p300 inhibitor curcumin were from Sigma (USA). The JNK inhibitor SP600125 was from Cell Signaling Technology (USA). A fluorescent dye calcein-AM used for live cell imaging was from Invitrogen (USA). The antibodies against p65, p38, phospho-p38, p44/42 MAPK, phospho-p44/42 MAPK, JNK, phospho-JNK, GFAP, and NF-κB were from Cell Signaling Technology (USA). The AQP4 antibodies were from Chemicon (USA). CRH was obtained from Tocris (1151, UK), and CRHR1 antagonist, CP154,526, was kindly donated by the Pfizer company (USA). The complementary DNA (cDNA) sequences encoding rat AQP4 (NM_001142366) were synthesized (Sangon Biotech, Shanghai, China) and then inserted into pmRFP-C1 at the XhoI and EcoRI sites.

Song T.T., Bi Y.H., Gao Y.Q., Huang R., Hao K., Xu G., Tang J.W., Ma Z.Q., Kong F.P., Coote J.H., Chen X.Q, & Du J.Z. (2016). Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia. Journal of Neuroinflammation, 13, 63.

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Protein Expression Profiling of Time Point Day 21

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SDS-PAGE was performed on 10% polyacrylamide gels and samples from all groups of
time point day 21 were transferred to Immobilon-P PVDF membranes (Millipore,
0.45 μl pore size, Millipore Corporation, Billerica, MA, USA). Membranes were
probed with one of the following primary antibodies: phospho-JNK, JNK,
phospho-p44/42 MAPK, p44/42 MAPK, phospho-p38, p38, phospho-IκBα (Cell
Signaling, Danvers, MA, USA), VE-cadherin (Santa Cruz Biotechnology, Texas, USA)
and actin (Millipore, Temecula, CA, USA). Protein bands were detected by
chemiluminescence (Santa Cruz Biotechnology, Texas, USA). For quantification,
densitometric analysis was performed.

Nikitopoulou I., Manitsopoulos N., Kotanidou A., Tian X., Petrovic A., Magkou C., Ninou I., Aidinis V., Schermuly R.T., Kosanovic D, & Orfanos S.E. (2019). Orotracheal treprostinil administration attenuates bleomycin-induced lung injury, vascular remodeling, and fibrosis in mice. Pulmonary Circulation, 9(4), 2045894019881954.

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Western Blot Analysis of MAPK Signaling

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Cultured HEK 293 cells were lysed with RIPA buffer (Sigma, St. Louis, MO, R0278) with protease inhibitor cocktail (Sigma, P8340), phosphatase inhibitor cocktail 2 (Sigma, P5726), phosphatase inhibitor cocktail 3 (Sigma, P0044). Protein samples (10 μg/well) were loaded to NuPAGE Novex 4–12% Bis-Tris protein gel (ThermoFisher Scientific, Carlsbad, CA, NP0336BOX) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat milk at room temperature for 1 hour and then probed with primary antibodies (phospho-p44/42 MAPK, Cell Signaling 9101, 1:4000, dilution) at 4°C overnight. Membranes were then incubated with HRP-conjugated secondary antibodies (goat anti-rabbit HRP, Bio Rad, 170-6515, 1:5000) for 1 hour and developed with SuperSignal solutions (Thermo Scientific). Then the membrane was stripped and probed again with primary antibodies (p44/42 MAPK, Cell Signaling 9102, 1:4000 dilution, β-actin 1:10,000, A5316, Sigma-Aldrich) and secondary antibodies including goat anti-mouse HRP (Bio Rad, 170-6516, 1:10000) and goat anti-rabbit HRP (Bio Rad, 170-6515, 1:5000),

Shen Y., Zhou M., Cai D., Filho D.A., Fernandes G., Cai Y., de Sousa A.F., Tian M., Kim N., Lee J., Necula D., Zhou C., Li S., Salinas S., Liu A., Kang X., Kamata M., Lavi A., Huang S., Silva T., Do Heo W, & Silva A.J. (2022). CCR5 closes the temporal window for memory linking. Nature, 606(7912), 146-152.

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Propionic Acid Regulates Cellular Signaling

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Primary antibodies: E-cadherin (Cell Signaling Technologies #3195), total YAP1 (Santa Cruz #G2710), Phospho-YAP1 S127 (Cell Signaling Technologies #4911S), LATS1 (Cell Signaling Technologies #3477S), p44/42 MAPK (Cell Signaling Technologies #9102), Phospho-p44/42 MAPK (Cell Signaling Technologies #9101), β-actin antibody (Sigma #A 5316), and V5 tag antibody (ThermoFisher Scientific #R960-25). Secondary antibodies: Horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling Technologies #7074) and anti-mouse (Cell Signaling Technologies #7076), Alexa Fluor 594 (ThermoFisher Scientific #A11005) and Alexa Fluor 488 phalloidin (ThermoFisher Scientific #A12379).
Reagents: Propionate (propionic acid # 402907) from Sigma-Aldrich Corporation.

Thirunavukkarasan M., Wang C., Rao A., Hind T., Teo Y.R., Siddiquee A.A., Goghari M.A., Kumar A.P, & Herr D.R. (2017). Short-chain fatty acid receptors inhibit invasive phenotypes in breast cancer cells. PLoS ONE, 12(10), e0186334.

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Quantitative Analysis of MAPK Signaling Pathways

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Western blots were measured as previously described (Huang and Chen 2013 (link)). Briefly, active and total form of ERK1/2, JNK and p38 MAPK accumulation were detected by Western blot analysis with using the following antigenes: phospho-P44/42MAPK (ERK 1/2) Thr²⁰²–Tyr²⁰⁴ (1:2000), phospho-SAPK/JNK Thr¹⁸³–Tyr¹⁸⁵ (1:1000), phospho-p38 MAPK (Thr¹⁸⁰–Tyr¹⁸²) (1:1000) (Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH (Abmart, Shanghai, China), and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000 dilution, Santa Cruz). Bands were visualized by means of enhanced chemiluminescence. Then NIH Image J program was used to quantify the results after scanning.

Zhang C.C., Gu W.L., Wu X.M., Li Y.M., Chen C.X, & Huang X.Y. (2016). Active components from Radix Scrophulariae inhibits the ventricular remodeling induced by hypertension in rats. SpringerPlus, 5, 358.

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