c-MET, HGF, and HGF/c-MET complexes in human carcinomas.

<p>A. NSCLC. B. Gastric carcinoma. C. Head and Neck carcinoma. c-MET, HGF, and HGF/c-MET assays were performed for all FFPE samples. Samples were rank ordered from low to high levels of HGF. The levels of c-MET receptor and HGF/c-MET were also tabulated. Red color-High ≥90^th percentile; Pink color-Low ≤10^th percentile. NSCLC and Gastric tumor exceeded 70% tumor content by pathologic analysis. In Head & Neck carcinoma samples, non-tumor tissue was removed by macrodissection. Di. c-MET was immunoprecipitated from NSCLC lysate samples and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Dii. c-MET was immunoprecipitated from gastric tumor lysates and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Samples that were c-MET phosphorylation (pY1003) positive (+) or c-MET (pY1003) negative (−) are summarized in Fig. A, B.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells.</p

Measurement of the HGF/c-MET complex by Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE) assay.

<p>A. SPPICE schemetic. c-MET was chemically cross-linked to expgenous HGF using the membrane impermeable sulfo-EGS cross-linker. Membrane proteins were solublized and HGF/c-MET complexes were measured using the SPPICE procedure (Bi) or by immunoprecipitation with HGF antibodies followed by Western blotting with anti-c-MET antibodies (Bii). C. c-MET and endogenous HGF in glioma cells were cross-linked as described above and SPPICE was used to measure HGF/c-MET complexes.</p

VeraTag FFPE c-MET quantification and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. Quantification of c-MET expression in FFPE preparations of H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, U87, and H661 FFPE cell lines using the VeraTag assay. Background signals generated using an isotype control IgG (shown in inset) were subtracted from the c-MET assay signals. B. Western blot analysis of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. β-actin levels in the blot serves as a loading control. C. ELISA measurements of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. ND: Not detectable. D. IHC detection of c-MET expression in FFPE preparations of H441, H226, MCF7, Ln18, U118, and H661 cell lines.</p

Antibody proximity VeraTag assay.

<p>A. Assay work-flow. B. Antibody proximity assay design for quantification of receptor and ligand-receptor complex.</p

VeraTag FFPE quantification of HGF/c-MET ligand-receptor complexes and correlation with c-MET (Tyr1003) phosphorylation.

<p>Ai. Schematic of the HGF/c-MET VeraTag FFPE proximity assay. Aii. Quantification of HGF/c-MET levels in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF/c-MET assay signals. B. VeraTag quantification of HGF/c-MET ligand receptor complexes in H661, MCF7, HGF stimulated MCF7, Ln18, Ln229, U138, U118, U87MG cell lines. C. Immunoprecipitation/Western blot analysis of c-MET (Tyr1003) phosphorylation and c-MET in Ln18, Ln229, U138, U118, and U87 MG glioma cells. The H596 cell line with exon 14 (L964-D1010) deletion was included as a negative control for c-MET (Tyr1003) phosphorylation.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA, and IHC in human tumor specimens.

<p>A. VeraTag FFPE quantification of HGF in NSCLC tumors. Isotype IgG control signals (inset) were subtracted from HGF signals. B. Immunoprecipitation/Western blot analysis of HGF in corresponding NSCLC tumor lysates. C. ELISA determinations of HGF in corresponding NSCLC specimens. D. IHC detection in NSCLC specimens.</p

c-MET quantification using the VeraTag FFPE assay and correlation with Western blot, ELISA and IHC in human tumor specimens.

<p>A. Quantification of c-MET in NSCLC specimens using the VeraTag FFPE proximity assay. Isotype IgG antibody signal (inset) was subtracted from c-MET assay signals. B. Western blot analysis of c-MET in corresponding NSCLC tumor lysates. β-actin levels in the blot serves as a loading control. Ci. ELISA determinations of c-MET levels in corresponding NSCLC tumor lysates. Cii. Correlation of c-MET measurements in NSCLC specimens by proximity assay and ELISA. Di. c-MET detection in NSCLC by IHC. Dii. Correlation of c-MET measurements in NSCLC by FFPE VeraTag proximity assay and IHC. Histologic score (H-score)  =  %IHC3⁺×3 + %IHC2⁺×2 + %IHC1⁺×1 + %IHC0×0 was calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015932#pone.0015932-Shi1" target="_blank">[10]</a>. Spearmen r and p values were calculated using graphpad prism software.</p

Epitope mapping of c-MET antibodies by peptide scanning.

<p>A. Peptide ELISA. A series of 43 overlapping peptides, each 14–15 amino-acid residues in length, spanning the complete intracellular domain of c-MET (K955-S1370), were evaluated to map the binding epitope of c-MET antibodies. Peptides were spotted on microtiter plates and ELISA assays were performed using standard protocols. The binding properties of peptide 28 and 43 localized the binding sites of c-MET antibody 3D4 and c-MET antibodies CVD13 and SP44, respectively. The sequence of peptide #28 and #43 are shown (inset). B. A schematic representing the c-MET receptor and binding sites for c-MET antibodies.</p

Supplemental Figure 2 from MMP-1 and Pro-MMP-10 as Potential Urinary Pharmacodynamic Biomarkers of FGFR3-Targeted Therapy in Patients with Bladder Cancer

FGFR3 siRNA and R3Mab diminish MMP-1 and MMP-10 mRNA leves in UMUC-14 cells.</p