This was quant itat ive ly transferred to th in layer plates and deve loped in chloroformmethanol. Specific activity was expressed as PM of product formed per microgram of prote in per hour.CDS cells were ma in ta ined in DMEM supp lemen ted wi th FCS.The transfection of CDS cells was performed wi th diethy lam inoethy ldex trane. After wash ing two timeswi th PBS, subconfluent CDS cells were <a href="http://www.targetmol.com/compound/GS-7340">Targetmol's
Tenofovir alafenamide</a> incubated wi th pg recombinant DNA in pg mldie thy lam i noethyldextrane in P B S for nun at C.E ightmillili te rs of DMEM supp lemen ted wi th FCS and PM ch loroqu ine was added, and the cells were incubated for. min at ream temperature.Med ium was changed, and cells were incubated in ml DMEM supp lemen ted wi th FCS for hat C.Probes were hea ted a longwi th sod ium dodecyl sulfate samp le buffer at C for min. After electrophoresis, the gelwas equil ib ra ted in transfer buffer for min. The transfer of prote in to an immobilon membrane was performed wi th a constant vo ltage of V for hon ice.The membrane was blocked in B S A in P B S for h, washed wi th PBS, and incubated overnight wi th a rabb it po lyc lonal ant ibody di lu ted, in. On morn ing the experiment, rats were separated in to three groups adm in istered ipwi th furosemide mgkg dissolved in DMSO, deltrasol mgkg, or a comb ination of the two compounds.The organic phase was transferred a fresh tube, evaporated under a stream ofnitrogen and dissolved of the mobile phase.One hundred microliters were in jec ted normalphase silica co lumn us ing a mobile phase consisting chloride, tetrahydrofuran, glac ial acetic acid. The flow ml m in, and the detect ion of the steroids was monitored detect ion limit of predn iso lone and prednisone was rig ml or mg of tissue.Constants were calculated by unwe igh ted linear regression ana l ysiswi th mean values of at least three experiments.As there is some variation at the in tercept a small, noncompetitive effect might in addition ac count for the inhibitory effect.As the cDNA of lmHSD an active protein that catalyzes both the oxidation and the reduction reactions, we tested furosemide as an inhibitor of EOHSD in both directions. The concentration of furosemide required to inhibit the enzyme activity by was about PM, respectively.Kivalues were of the same magnitude as those derived from the studies using liver microsomes.Only the active principle of licorice, glycyrrhetinic acid, had a similar inhib itory effect on lpOHSD activity.Corticosterone concentrations are given in PM, and the rate was defined as micromole of substrate oxydized per microgram of protein per hour.A similar effect of furosemide was observed in the present study when liver microsomes were used as a source of OHSD, indicating that the effect is not restricted to the kidney.To define more precisely the enzyme involved, we trans fected CO cells with the rat clone of the lljOHSD and assessed the effect of furosemide on the activity of that enzyme.The wild type of COS cells is virtually unable to convert hydroxy to the corresponding llketo steroids and vice uersu.