Fig 1: Detection of functional NAPRT by 3C6D2 in human tumor cell lines(A) NAPRT expression levels were determined in a panel of cancer cell lines by immunoblotting using monoclonal antibody 3C6D2 or the polyclonal antibody HPA023739. Tubulin staining was used as a protein loading control. (B) Functional NAPRT activity was assessed in cells treated with a lethal dose of the NAMPT inhibitor GMX1778 (50 nM) in the presence or absence of exogenous niacin (1 μM) for 72 h. Viability was determined using the Cell-Titer Glo assay and is expressed as the mean percentage viability relative to cells treated without GMX1778. Error bars represent the SD (n=6 replicates).
Fig 2: Monoclonal antibody 3C6D2 is highly specific for NAPRT proteinH1299 cells were transfected with either a non-targeting siRNA (siNT), an NAPRT-targeting siRNA (siNAPRT) as indicated for 72 h and cell lysates harvested. 10 μg of whole cell lysates from siRNA transfected cells or 2 ng of purified NAPRT immunization fragment (aa 256-515) or recombinant GST-NAPRT were resolved on BioRad stain-free polyacrylamide gels and detected by immunoblotting with (A) the 3C6D2 monoclonal antibody or (B) a commercially available polyclonal NAPRT specific antibody (HPA023739). Total protein was visualized on the protein containing nitrocellulose membrane using a fluorescence detection imager.
Fig 3: Frequency of NAPRT staining in human lung and brain cancers(A) A representative comparison of NAPRT expression levels in human tumor-adjacent normal brain tissue (upper left) and glioblastoma tumor tissue (upper right) or in normal lung (lower left) compared to small cell lung carcinoma (lower right). Each sample was stained with 3C6D2 and CAT hematoxylin. Scale bars: 100 μm (brain) and 40 μm (lung). (B) Lung or (C) brain cancer tumor micro arrays (TMAs) were stained with 3C6D2/CAT hematoxylin by IHC to determine expression levels of NAPRT protein. The frequency NAPRT positive cancerous cells within the tumor sample was scored as 0-10 % (white), 11-90 % (grey) or 91-100 % (black) positively stained. Samples scored as equivocal (hatched bars) could not be evaluated. The results are indicated as the percentage of total cases examined within a tumor sub-type. n = the number of different tumor cases evaluated.
Fig 4: Immuno-histochemical detection of NAPRT expression from FFPE human xenograft, histology and cell line samples(A) NAPRT expression in three FFPE embedded xenograft tumors from cell lines: H929 (multiple myeloma), A549 (non-small cell lung) and HT29 (colon). Xenograft slices were stained with 3C6D2 and CAT hematoxylin. Scale bars represent 200 μm. (B and C) Examples of NAPRT expression from FFPE human tumor samples from (B) ovarian cancer and (C) lymphoma. Adjacent slices of tumor tissue were stained with 3C6D2 and CAT hematoxylin counter stain (left) and hematoxylin and eosin staining (H&E) (right). Scale bars represent 60 μm (lymphoma) or 200 μm (ovarian cancer). (D) FFPE cell pellets from NAPRT positive HT29 and NAPRT negative HT1080 cells were stained with the concentration range of various commercially available NAPRT monoclonal antibodies as indicated and CAT hematoxylin counter stained. IgG1 and IgG2α were used as a negative staining control.
Fig 5: Epitope mapping of 3C6D2 antibody by ELISA and IHC staining with epitope peptide competition(A) ELISA assay reactivity for four peptides near the 3C6D2 antibody epitope on NAPRT. OD, optical density (B) ICC staining of HT-29 cell pellets with 3C6D2 in the presence or absence of a 200-fold molar excess of epitope containing peptides as indicated. Scale bars, 200 μm.
Supplier Page from MilliporeSigma for Anti-NAPRT antibody produced in rabbit