Egfr d38b1 rabbit mab

For each case, FFPE samples were assayed for EGFR, HGF, total and phosphorylated MET using the following antibodies: EGFR (D38B1) rabbit mAb (Cell Signaling, USA), HGF (4C12.1) mouse mAb (Millipore, USA), MET (SP44) mouse mAb (Ventana Medical Systems, USA), and p-MET Y1234/1235 (3D7) rabbit mAb (Cell Signaling). Immunostaining was performed as described previously [34 (link)]. As a positive control, sections of NSCLC tumors with known marker expression were stained. Sections from the same specimens incubated with normal mouse and rabbit IgG2 instead of primary antibodies were used as negative controls. Antigen preservation in tissues was confirmed by assaying sections from the same tissue array for expression of phospho-tyrosines, using an anti-phosphotyrosine mAb (4G10, Millipore).
Stainings were evaluated by two pathologists (F.R. and E.G.). HGF was evaluated in tumoral stroma; EGFR, MET and p-MET were quantified in the membrane of tumor cells. In addition, a semiquantitative histoscore (Hscore) was calculated by estimation of the percentage of tumor cells positively stained with low, medium, or high staining intensity after applying a weighting factor to each estimate. The formula used was Hscore = (low %) × 1 + (medium %) × 2 + (high %) × 3, and results ranged from 0 to 300.

To evaluate knockdown efficiency, cells grown on six-well dishes, transfected with the indicated siRNAs and washed with ice-cold PBS. In brief, cells were lysed with 200 μL of RIPA buffer (Thermo Scientific, 89900) supplemented with 5 mM EDTA, and 1X protease inhibitor (Thermo Scientific, 87786) for 1 h on ice. Lysates were centrifuged at 12,000×g for 15 min. Proteins in supernatants were quantified using the Pierce BSA protein assay kit (Thermo Scientific, 23227) and separated by SDS-PAGE. Then, samples were electrotransferred onto polyvinylidene difluoride membranes. Immunoblotting was performed using the following dilutions of primary antibodies and secondary antibodies: 1:8000 EGFR (D38B1) rabbit mAb (Cell Signaling, 4267); 1:4000 β5-integrin (D24A5) rabbit mAb (Cell Signaling, 3629); 1:4000 Src (14H2L20) rabbit mAb (Thermo Scientific, 701396); 1:5000 GAPDH (D16H11) rabbit mAb (HRP conjugated) (Cell Signaling, 8884); 1:30,000 IgG Fraction Monoclonal Mouse Anti-Rabbit IgG (HRP conjugated (Jackson ImmunoResearch, 211-032-171). Chemoluminescence signal was developed using Amersham ECL Prime Western Blotting detection reagents (GE Healthcare, RPN2232) and images were obtained with a ChemiDoc XRS + system (BioRad).