Nitric oxide (NO) has a broad range of biological activities and is implicated in signaling pathways in phylogenetically diverse species. Nitric oxide synthases (NOS) are the enzymes responsible for synthesis of NO and several distinct NOS isoforms are produced from three distinct genes. The inducible form of NOS, iNOS (also designated NOS-II), is Ca2+ independent and is expressed in a broad range of cell types. This form of NOS is induced in macrophages and monocytes after stimulation with cytokines and exposure to microbial products, such as LPS.
Confluent cultures of mouse macrophage cells (J774A.1) were treated with LPS (1 μg/ml) for 18 hrs at 37°C (cat. # ML8741), then cells were treated with pervanadate (1 mM) for 30 min. at 37°C (cat. # ML8751). Cells were lysed in 1% SDS, 1.0 mM sodium ortho-vanadate, 1 mM sodium fluoride, 10 mM Tris (pH 7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer. The pervanadate treated lysate has increased tyrosine phosphorylation of many proteins in J774A.1 as demonstrated using anti-Phosphotyrosine antibody (PP2221).
Xie, Q.W. et al. (1992) Science 256:225.
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*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blot mobilities of known proteins with similar MW.
This kit contains: