P met y1234 1235

Manufactured by Cell Signaling Technology

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P-Met (Y1234/1235) is a rabbit monoclonal antibody that recognizes c-Met protein phosphorylated at tyrosine residues 1234 and 1235. This antibody is designed for use in Western blotting applications to detect the activated, phosphorylated form of c-Met.

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P-Met (37 citations) Anti-p-MET Y1234/1235 (3 citations) Y1234/1235 (2 citations)

6 protocols using p met y1234 1235

Western Blotting for Protein Quantification

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Western blotting was performed using standard methods. After treatment with indicated drugs, cells were washed with cold PBS and lysed in the following lysis buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 50 mM NaF, 10 nM β-glycerophosphate, 1 mM sodium vanadate, 0.5 mM DTT, 4 μg/mL leupeptin, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 16,000 × g for 5 min at 4°C. Protein concentrations were determined by BCA assay (Thermo Scientific). Proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham). Immunoblotting was performed per antibody manufacturer's specifications. Antibodies for MET, P-MET (Y1234/1235) HER2, P-HER2 (Y1221/1222), P-AKT (S473), and P-S6 (S240/244), (Cell Signaling) were used at 1:1000 dilution; P-ERK (Cell Signaling) was used at 1:2000 dilution. GAPDH (Millipore) was used at 1:1000 dilution. Protein detection on Western blots was performed using SuperSignal chemiluminescence (Thermo Scientific).

Kwak E.L., Ahronian L.G., Siravegna G., Mussolin B., Godfrey J.T., Clark J.W., Blaszkowsky L.S., Ryan D.P., Lennerz J.K., Iafrate A.J., Bardelli A., Hong T.S, & Corcoran R.B. (2015). Molecular heterogeneity and receptor co-amplification drive resistance to targeted therapy in MET-amplified esophagogastric cancer. Cancer discovery, 5(12), 1271-1281.

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Investigating Receptor Tyrosine Kinase Signaling

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Reagents were: Insulin (Sigma, I0516), NEAA (Gibco, 11140050), gentamycin (Sigma, G1272), G418 (OZ Biosciences, GS21000).
Inhibitors were: BYL719 (Selleckchem, S2814), SHP099 (Selleckchem, S8278), GDC-0941 (Selleckchem, S1065), GS-493 (Millipore, 538099). All inhibitors were solved in DMSO.
Growth factors were: NRG1 (396-HB-050), EGF (236-EG-200) and FGF10 (345-FG-025), purchased from R&D Systems.
Antibodies: AKT (Cell Signaling, 9272), p-AKT S473 (Cell Signaling, 4060), p-4E-BP1 T37/46 (Cell Signaling, 2855), ERK (Cell Signaling, 9102), p-HER3 Y1289 (Cell Signaling, 4791), p-MET Y1234/1235 (Cell Signaling, 3077), p-FGFR Y653/654 (Cell Signaling 3471), p-S6 ribosomal protein S240/244 (Cell Signaling, 5364), p-ERK Y204 (Santa Cruz, sc-7383), HSP90 (Santa Cruz, sc-13119), SHP2 (Santa Cruz, sc-280), p-SHP2 Y542 antibody (AbCam, 62322), p-EGFR Y1068 (AbCam, 5644). Secondary rabbit (#111-035-144) and mouse (#115-035-062) antibodies were from Dianova.

Heynen G.J., Lisek K., Vogel R., Wulf-Goldenberg A., Alcaniz J., Montaudon E., Marangoni E, & Birchmeier W. (2022). Targeting SHP2 phosphatase in breast cancer overcomes RTK-mediated resistance to PI3K inhibitors. Breast Cancer Research : BCR, 24, 23.

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Immunoblotting of Receptor Tyrosine Kinases

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Whole cell extracts were prepared by scraping cells in lysis buffer (150 mM NaCl, 5 mM EDTA, 0.5% NP40, 50 mM Tris, pH 7.5), resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies to p-EGFR (Y1068), EGFR, p-ErbB2 (Y1221), ErbB2, p-ErbB (Y1289), ErbB3, p-IGF1R (Y1135), IGF1R, p-PDGFRβ (Y751), PDGFRβ, p-Met (Y1234/1235), Met, p-AKT (S473), pan AKT, p-ERK (T202/204), and ERK were from Cell Signaling (Danvers, MA, USA); Phospho-IRS1 (Y612) was from EMD Millipore (Burlington, MA, USA). IRS1 was from Bethyl Laboratories (Montgomery, TX, USA). PRCP was from R&D systems (Minneapolis, MN, USA). β-actin was from Santa Cruz (Dallas, TX, USA). Primary antibodies were detected with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Life Technologies, Carlsbad, CA, USA), using Clarity chemiluminescence (BIO-RAD, Hercules, CA, USA). For some blots, PVDF membranes were cut into halves at the protein markers around 75–100 KD. The upper parts were immunoblotted for high molecular weight proteins, and the lower parts were immunoblotted for low molecular weight proteins.

Duan L., Calhoun S., Perez R.E., Macias V., Mir F., Pergande M.R., Gattuso P., Borgia J.A, & Maki C.G. (2022). Prolyl Carboxypeptidase Maintains Receptor Tyrosine Kinase Signaling and Is a Potential Therapeutic Target in Triple Negative Breast Cancer. Cancers, 14(3), 739.

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Signaling Pathway Protein Analysis

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The specific antibody of EGFR, p-EGFR (Tyr1068), p-PI3K, Akt, mTOR, p-mTOR (Ser2448), Met, p-Met (Y1234/1235), ERK1/2, p-ERK1/2, β-actin and β-tubulin were purchased from Cell Signaling Technology (Beverly, MA). PI3K and p-Akt (Ser473) were obtained from ABcam (Cambridge, UK). The enhanced chemiluminescence (ECL) system was from Millipore (Millipore, MA, USA). Epidermal growth factor (EGF) was purchased from Biosource International Inc. (Camarillo, CA) and dissolved in phosphate-buffered saline (PBS).

Han S.Y., Ding H.R., Zhao W., Teng F, & Li P.P. (2014). Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR. BMC Complementary and Alternative Medicine, 14, 165.

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Western Blot Analysis of Signaling Pathways

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Cells were treated with LY3009120 or DMSO as indicated and prepared for Western blotting as previously described [52 (link)]. Signal was detected by enhanced chemiluminescence and visualized on the ChemiDoc XRS instrument (Bio-Rad). Antibodies against ARAF (#4432), pBRAF S445 (#2696), pCRAF S338 (#9427), pEGFR Y1068 (#3777), EGFR (#4267), pRSK T359 (#8753), RSK1 (#9333), pMEK1/2 S217/S221 (#9154), pERK1/2 T202/Y204 (#4370), ERK1/2 (# 4696), pS6 S240/244 (“pS6”; #5364), rpS6 (“S6”; #2317), pAKT S473 (#4060), AKT (#2920), Rb (#9309), pMET Y1234/1235 (#3077), MET (#8198) (all from Cell Signaling Technology), BRAF (Santa Cruz Biotechnology, Inc. #sc-9002), CRAF (Bethyl Labs #A301-519A), pRb S780 (BD Biosciences #558385), and α-Tubulin (Sigma #T5168) were diluted in 5% BSA in 1x TBS-T.

Vakana E., Pratt S., Blosser W., Dowless M., Simpson N., Yuan X.J., Jaken S., Manro J., Stephens J., Zhang Y., Huber L., Peng S.B, & Stancato L.F. (2016). LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer. Oncotarget, 8(6), 9251-9266.

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Molecular Profiling of Mutant Melanoma Cell Lines

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BRAF and NRAS mutant melanoma cell lines were obtained from various sources (Supplementary Methods). The tetracycline (Tet)-HGF-G361 cell line was generated as described (Supplementary Methods). Cell lines were maintained in vendor- or investigator-recommended media. Vemurafenib was purchased from Jubilant Life Sciences; the BRAF inhibitor C-1 [35 (link), 38 (link)], dabrafenib, trametinib, the MET inhibitors AMG 337 and Compound 20 (an AMG 337 analog), the PI3K inhibitor AMG 511, and the MEK inhibitor PD0325901 were supplied by Amgen Inc. Antibodies against pMEK (S217/221), pAKT (S473), pMET (Y1234/1235), pGAB1 (Y627), MET, GRB2, SOS1, ERBB3, EGFR, IGF1R, FGFR1, IRS1, and PDGFRB were obtained from Cell Signaling Technology. Antibodies against pERK (T185/Y187; Life Technologies), GAB1 (Millipore), and beta-actin (Abcam) were also obtained.

Caenepeel S., Cooke K., Wadsworth S., Huang G., Robert L., Moreno B.H., Parisi G., Cajulis E., Kendall R., Beltran P., Ribas A., Coxon A, & Hughes P.E. (2017). MAPK pathway inhibition induces MET and GAB1 levels, priming BRAF mutant melanoma for rescue by hepatocyte growth factor. Oncotarget, 8(11), 17795-17809.

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