In p53 protein immunoprecipitated from A549 cells exposed to NaVO3, the marked phosphorylation was identified at only Ser15, plus the faint phosphorylation was found at Ser37, Ser46 and Ser392 . About the other hand, clear phosphorylation was not observed at Ser6, Ser9 and Ser20 in p53 immunoprecipitated, whereas equal quantities of p53 protein have been detected . Effects of vanadate compounds exposure on p53 phosphorylation at Ser15 Additionally to NaVO3, the exposures to 100 ?MNa3VO4 and NH4VO3 for 48 h, both compounds are pentavalent species, induced the accumulation of p53 phosphorylated at Ser15 and p53 protein without having the alteration of actin level in A549 cells . Results of U0126 on NaVO3-induced p53 phosphorylation at Ser15 In response to NaVO3 exposure, the degree of phosphorylated kind of extracellular signal-regulated protein kinase greater following 1 h and remained elevated even at 48 h, whereas that of total ERK was not changed through the incubation time period of 48 h .
Treatment method with U0126 , an inhibitor of each activated and non-activated forms of MAPK/ERK kinase , suppressed NaVO3-induced phosphorylation of ERK in a dose-dependent method . Nevertheless, Dovitinib TKI258 NaVO3-induced Ser15 phosphorylation and accumulation of p53 protein were not suppressed by therapy with U0126. Alternatively, a higher dose of U0126 enhanced slightly NaVO3-induced Ser15 phosphorylation . Effects of wortmannin or caffeine on NaVO3-induced p53 phosphorylation at Ser15 Treatment method with wortmannin , an inhibitor of DNA-activated protein kinase and ATM , suppressed NaVO3-induced Ser15 phosphorylation in the concentrations of a hundred and 200 nM . The accumulation of p53 protein was suppressed by a increased concentration of wortmannin .
Moreover, treatment method with caffeine , an inhibitor of ATM and ATM-Rad3-related protein , suppressed NaVO3-induced Ser15 phosphorylation and accumulation of p53 protein considerably depending on the concentration . To the other hand, the levels of actin were not affected selleckchem TAK-285 by remedy with wortmannin or caffeine . These suppressive effects of wortmannin and caffeine propose that ATM could be liable for NaVO3- induced p53 phosphorylation at Ser15 in A549 cells. Results of ATM knockdown on NaVO3-induced p53 phosphorylation at Ser15 Transfection with siRNA targeted towards the human ATM gene abolished essentially completely the expression of ATM protein in each A549 cells taken care of devoid of and with NaVO3 . In cells transfected with siRNA, NaVO3-induced Ser15 phosphorylation and accumulation of p53 protein have been suppressed by 69% and 39%, respectively .
No substantial change from the degree of actin was identified in cells transfected with siRNA followed by NaVO3 publicity.