37-0100

antibody from Invitrogen Antibodies

Targeting: MET DFNB97, HGFR, RCCP2

Provider product page for 37-0100

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [24]
Comments [0]
Validations
Immunohistochemistry [1]
Other assay [11]

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Product number: 37-0100 - Provider product page
Provider: Invitrogen Antibodies
Product name: c-Met Monoclonal Antibody (3D4)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This antibody is specific for the cytoplasmic domain of the c-Met protein. On western blotting, this antibody recognizes a band at approximately 145 kDa. During development reactivity was confirmed with human HT-29 breast carcinoma lysates and c-Met cytoplasmic domain recombinant protein by both western blot, and with formalin-fixed, paraffin-embedded breast carcinoma tissues by immunohistochemistry.
Antibody clone number: 3D4
Concentration: 0.5 mg/mL

MET protein structure - 37-0100 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Classification of 56 patients by prognosis based on 108 protein expression values. (A) Boxplot of protein expression values before (left) and after (right) quantile normalization. The plot shows the quantile normalized distribution of protein expression values for each patient. Horizontal axis represents individual patients. Vertical axis represents H-score. (B) Heatmap and dendrogram as a result of hierarchical clustering of GBM samples. Top dendrogram represents clustering of patients. Left dendrogram represents clustering of proteins. Of two patient branches, samples in the left branch represent cluster 1 and samples in the right branch represent cluster 2, consisting of 19 and 37 patients, respectively. (C) Univariate survival analysis of overall survival by Kaplan-Meier method. Kaplan-Meier survival plot of the two clusters of patients defined by the hierarchical clustering. Cluster 1 is the poor survival group. The log-rank test shows that the difference between two curves is significant (p

Supportive validation

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Experimental details: Fig. 6 Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen (r) assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen (r) donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF),

Supportive validation

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Experimental details: Figure S5. MDCK cell morphogenesis assay and signalling pathway activation using K1K1, K1K1S2, and K1K1S4 mutants. (A) MDCK cell (~500) were seeded into a thick layer of a 1:1 collagen/Growth Factor Reduced Matrigel and treated with semi-log dilutions (100 pM to 300 nM) of hepatocyte growth factor/scatter factor, K1K1, K1K1S2, and K1K1S4 twice a week for 1 mo. The cells were fixed and stained with Evans blue and DAPI. The image of the full plate is presented. (B) Colonies were observed in bright field using inverted microscope observed 4x objective on a Nikon Eclipse TS100 microscope. (C) Phosphorylation analysis of MET signalling pathway by Western blot on HeLa cell lysates after stimulation with semi-log dilution (0.3-30 nM) of K1K1, K1K1S2, or K1KS4 for 10 min. Loading controls are based on total MET, total Akt, and total ERK present in each lane. Source data are available for this figure. Source Data for Figure S5

Supportive validation

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Experimental details: Resistance is not due to loss of cell responsiveness to MV-DN30. (A) Western blot of MET ECD in cell supernatants of EBC1 WT, R20 and R80 cells in the absence or in the presence of MV-DN30 for 24 h. (B) Western blot revealing MET protein (top) and MET tyrosine phosphorylation (Y1234-Y1235) (middle) levels in EBC1 WT, R20 and R80 cells grown either in the absence (24 h deprivation) or in the presence (24 h) of MV-DN30. Vinculin (bottom) was used as loading control. (C) Box-plot of Fluorescence intensity of plasma-membrane bound MET (A.U. : Arbitrary Units) in EBC1 WT, R20 and R80 cells, either in the absence (24 h deprivation) or in the presence (24 h) of MV-DN30. Plasma-membrane staining as in Fig. 1E. (D) Western blot of MET ECD (top) and total MET protein levels (middle) in EBC1 WT, R20 and R80 cells upon MV-DN30 treatment (24 h). Resistant cells were antibody-deprivated for 24 h before treatment. Vinculin (bottom) was used as loading control. (E) Immunoprecipitation assays of MET (from cell lysates, top) and of MET ECD (from cell supernatants, bottom) upon Pulse-Chase metabolic protein labeling in EBC1 WT and R80 cells, in the absence or in the presence of MV-DN30. (F) Viability of EBC1 WT (left graph) and R20 cells (right graph) in the presence of increasing concentrations of MV-DN30. Results are normalized to WT untreated cells (100%) (left graph) or to R20 cells treated with 20 mug/mL (100%) (right graph) +- s.d. (***P < 0.001, *P < 0.05).

Supportive validation

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Experimental details: MV-DN30 resistant cells display increased MET synthesis. (A) Real-Time qPCR analysis of MET gene copy number in EBC1 WT, R20 and R80 cells. MET copy number +- s.d. (**P < 0.01; ***P < 0.001) was normalized to diploid control cells (HEK-293T and A549 cells). (B) Real-time qPCR of MET mRNA expression level in EBC1 WT, R20 and R80. (C) Real-time qPCR of MET mRNA level in EBC1 WT cells transduced with a control mock vector or with two different doses (MET++ : white oblique lines and MET+++: white squares) of a MET encoding lentiviral vector and in R20 and R80 cells transduced with the control vector alone (mock). (B, C) MET mRNA fold increase +- s.d. (**P < 0.01; ***P < 0.001) was normalized to that of normo-expressing cells (HEK-293T and A549 cells). (D) Viability of EBC1 WT cells transduced with different amounts of MET cDNA or mock vector, kept in the presence or in the absence of MV-DN30, was evaluated 72 h after treatment. Results are normalized with the untreated cells (100%) +- s.d. (**P < 0.01; ***P < 0.001). (E) Western blot showing MET protein and MET Tyrosine phosphorylation levels of transduced WT, R20 and R80 EBC1 cells kept for 24 h in the presence or in the absence of the indicated MV-DN30 concentrations. A549 and HEK-293T cells were stimulated with HGF (100 ng/mL) as a comparison for physiological levels of MET phosphorylation.