Pmapk thr202 tyr204

Manufactured by Cell Signaling Technology

Sourced in China

PMAPK (Thr202/Tyr204) is a lab equipment product that detects and measures the phosphorylation of p44/42 MAPK (Erk1/2) at Thr202 and Tyr204. It is used to assess the activation state of this important cell signaling pathway.

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5 protocols using pmapk thr202 tyr204

Characterization of FLT3 Mutations

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DNA constructs and vectors were used as described before [9] (link), [25] (link). FLT3-I867S and FLT3-D839G constructs were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). Denotation: W78: ITD1, Npos: ITD2, W51: ITD3. Figure S1 displays the locations and insertions respectively substitutions of the analyzed mutations.
The following antibodies were used: FLT3 (S18) and goat anti-mouse secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). AKT, MAPK, pSTAT5 (Tyr694), pAKT (Ser473) and pMAPK (Thr202/Tyr204) all from Cell Signaling Technology (Danvers, MA). STAT5 from R&D Systems (Minneapolis, MN). β-actin and goat anti-rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO). CD-135-PE from Beckman Coulter (Brea, CA). IgG1 PE Isotype control from BD Pharmingen (BD Bioscience, Franklin Lakes, NJ).

Janke H., Pastore F., Schumacher D., Herold T., Hopfner K.P., Schneider S., Berdel W.E., Büchner T., Woermann B.J., Subklewe M., Bohlander S.K., Hiddemann W., Spiekermann K, & Polzer H. (2014). Activating FLT3 Mutants Show Distinct Gain-of-Function Phenotypes In Vitro and a Characteristic Signaling Pathway Profile Associated with Prognosis in Acute Myeloid Leukemia. PLoS ONE, 9(3), e89560.

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Protein Immunodetection and Analysis Protocol

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The following antibodies were used in this study: anti-Girdin (R&D Systems, Minneapolis, MN, #AF5345, for WB and IP), anti-4F2hc (H300, Santa Cruz Biotechnology, Santa Cruz, CA, for WB, clone MEM-108, Biolegend [#315602] for IP, and mouse monoclonal anti-4F2hc [clone HBJ 127] for IF), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology, #sc-459), anti-Myc (clone 9E10, Santa Cruz Biotechnology), anti-poly-histidine (clone HIS-1, Sigma, St. Louis, MO), anti-Flag (clone M2, Sigma), anti-β-actin (clone AC-74, Sigma), normal mouse IgG (Millipore, Milford, MA, #12–371), and normal sheep IgG (Millipore, #12–515). Antibodies to pS6K (Thr389) (#9205), S6K (#9202), S6 (#2217), pS6 (Ser240/244) (#2215), MAPK (#9102), pMAPK (Thr202/Tyr204) (#9106), Lamp1 (#9109), mTOR (#2983), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA). Flag M2 affinity gel, adenosine 5′-triphosphate (ATP), and amino acids were purchased from Sigma. Phos-tag Acrylamide (Wako, Saitama, Japan) was used for the generation of the Phos-tag gel to analyze protein phosphorylation in cells.

Weng L., Han Y.P., Enomoto A., Kitaura Y., Nagamori S., Kanai Y., Asai N., An J., Takagishi M., Asai M., Mii S., Masuko T., Shimomura Y, & Takahashi M. (2018). Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex. PLoS Biology, 16(3), e2005090.

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Signaling Pathway Protein Analysis

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Antibodies were purchased from Cell Signaling (Beverly, MA): anti- AKT (CS#4691), p-AKT (Ser473) (CS#9271), p-AKT (Thr308) (CS #4056), mTOR, p-mTOR (Ser2448) (CS#2971), PDK1 (CS#13037), p-PDK1(Tyr373/376) (bs-3017R), p-PDK1(Ser241) (CS#3061), MAPK (CS#4695), p-MAPK (Thr202/Tyr204) (CS#4370), PTEN (CS#9559), p-PTEN (CS#9549), YAP (CS#4912), p-YAP (s127) (CS#13008), p-YAP (s397) (CS#13619). Monoclonal anti-β-actin (Sigma#A5449) was purchased from Sigma-Aldrich (St Louis, MO).

Meraz I.M., Majidi M., Fang B., Meng F., Gao L., Shao R., Song R., Li F., Lissanu Y., Chen H., Ha M.J., Wang Q., Wang J., Shpall E., Jung S.Y., Haderk F., Gui P., Riess J.W., Olivas V., Bivona T.G, & Roth J.A. (2023). 3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib. Communications Biology, 6, 509.

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Investigating Cellular Signaling Pathways

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The following reagents were used: Tris, glycine, sodium dodecyl sulfide and bovine serum albumin (BSA) (Sigma). Antibodies included: p-MAPK^{Thr202/Tyr204}, p-AKT^Ser473, p-mTOR^Ser2448, p-S6K^Thr389 and p-4E-BP1^Thr37746 (Cell signaling Technology); β-actin and myogenin (Genetex); myoD1 (Proteintech); vimentin, cytokeratin 8, Ki-67 and α-SMA (Abcam) and desmin (Servicebio Co. Ltd., China). Experiments were performed under a project license (NO.: L201501027) granted by ethics board of Sun Yat-sen University, in compliance with all national and/or institutional guidelines for the care and use of animals.

Lai H., Guo Y., He W., Sun T., Ouyang L., Tian L., Li Y., Li X., You Z, & Yang G. (2020). Non-target genetic manipulation induces rhabdomyosarcoma in KrasPten-driven mouse model of ovarian cancer. Translational Cancer Research, 9(12), 7458-7468.

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Capmatinib Regulation of Met and Downstream Signaling

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Whole-cell lysates were collected in a RIPA buffer containing
protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Mouse
derived cell lines were grown to 90% confluency overnight by seeding plates with
50,000 – 75,000 cells. Cells were then serum starved for 24 hours and
then treated with capmatinib for 2 hours followed by 100ng/mL of HGF for 15
minutes. Cells were then washed with PBS and harvested in RIPA buffer plus
protease inhibitor cocktail (Roche). Lysates (20 μg) were resolved on a
4%−20% TGX SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane
(Invitrogen). After blocking for 1 hour with 5% dry milk in TBST buffer (20
mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20), blots were probed
overnight at 4°C with the following primary antibodies from Cell
Signaling Technology: Met (25H2; #3127), pMET (D26; #3077), AKT (#9272), pAKT
(S473; #9271), MAPK (#9102), pMAPK (Thr202/Tyr204; #9101), and β-tubulin
(#2146). Blots were reacted with peroxidase- conjugated antibody for 1 hour
(Cell Signaling Technology) and visualized using ECL detection (Amersham).

Peacock J.D., Pridgeon M.G., Tovar E.A., Essenburg C.J., Bowman M., Madaj Z., Koeman J., Boguslawski E.A., Grit J., Dodd R.D., Khachaturov V., Cardona D.M., Chen M., Kirsch D.G., Maina F., Dono R., Winn M.E., Graveel C.R, & Steensma M.R. (2018). Genomic status of MET potentiates sensitivity to MET and MEK inhibition in NF1-related malignant peripheral nerve sheath tumors. Cancer research, 78(13), 3672-3687.

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