Figure 1.
Polyclonal GSNOR antibodies react with other ADHs.
22.5 ng purified recombinant proteins were separated by SDS-PAGE, and immunoblots were performed to determine antibody reactivity to GSNOR, ADH IA, ADH IB, ADH II, and ADH IV. With the exception of ADH IA, the purified recombinant proteins were generated with a fusion protein tag during cloning, and the tag was cleaved off during purification as described in the Materials and Methods. The molecular weights of the fusion proteins are approximately 50–51 kDa, and the final, purified proteins are 39–41 kDa. Human GSNOR consistently migrates faster than the calculated molecular weight. As seen by for the presence of both 50 and 40 kDa bands for ADH II, the majority of the protein in this preparation still contains the fusion protein tag. However, the purified GSNOR protein used for immunization was confirmed to be full length, and free of additional tag sequence as described in the Materials and Methods. A) Commercially available rabbit polyclonal GSNOR antibody (Proteintech #11051-1-AP). B) In-house polyclonal antibody generated by immunization of rats with purified, recombinant, full length human GSNOR protein at Biomodels (Watertown, MA) for N30 Pharmaceuticals.
Figure 2.
Monoclonal GSNOR antibodies are specific for GSNOR.
Purified recombinant proteins were separated by SDS-PAGE, and immunoblots were performed to determine antibody reactivity to GSNOR, ADH IB, ADH II, and ADH IV. Three independent monoclonal antibodies were tested: A) N30-F6 mouse anti-GSNOR; B) N30-G11 mouse anti-GSNOR; C) N30-C3 rat anti-GSNOR.
Figure 3.
GSNOR is present in normal lung tissue.
Normal human lung tissue microarrays were stained with N30-C3 monoclonal GSNOR antibody (1 µg/mL) followed by DAB detection. Bronchial epithelial cells, alveolar macrophages, and type 2 pneumocytes in alveoli are strongly stained.
Figure 4.
GSNOR is found in human lung cancer tissues stained with monoclonal GSNOR antibodies.
Normal human lung cancer tissue microarrays were stained with monoclonal GSNOR antibodies (N30-C3, N30-F6, N30-G11) or a commercially available polyclonal GSNOR antibody (11051-1-AP) followed by DAB detection. Various human lung cancer tissues are strongly stained by all three GSNOR monoclonal antibodies, but staining is less prominent when the non-specific polyclonal GSNOR antibody is used.
Table 1.
Quantitation of GSNOR positive lung cancer tissue sections.
Figure 5.
GSNOR and ADH gene expression in lung cancer cDNA arrays.
GSNOR (gene name ADH5), ADH IB (ADHIB), ADH II (ADH4), and ADH IV (ADH7) mRNA levels were evaluated by qPCR in human lung cancer cDNA arrays (Origene, #HLRT504, lot #0411, Rockville, MD) and normalized to β-actin levels. Arrays contained 23 matched pairs of normal and cancerous lung tissue. Tumor expression relative to the matched normal sample was calculated using the ΔΔCt method. Relative quantities <1 represent decreased expression in the tumor sample. Lung cancer specimens included Stage IA (n = 4), IB (n = 4), IIA (n = 2), IIB (n = 8), IIIA (n = 3), and IIIB (n = 2) with each sample paired with adjacent normal tissue. No correlation between GSNOR mRNA levels and lung cancer was observed, while expression of ADH IB was strongly reduced in lung cancer samples.