Heme oxygenase-1 and ectonucleotidases functional assays.

<p>(<b>A</b>) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *<i>p</i><0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups.