TI - Diminished ERK1/2 activation in the nucleus by ESAT-6 was due to some tyrosine phosphatase . AB - To evaluate whether any phosphatase was involved in the dePHOSphorylation of ERK1/2 in the nucleus of ESAT-6 treated cells , RAW264.7 cells were stimulated with 5 mug/ml of ESAT-6 in presence of 1 mM sodium orthovanadate (Na3VO4) , which is a protein tyrosine phosphatase inhibitor [48] . We found that in the presence of Na3VO4 , pERK1/2 appeared in the nucleus ( Fig 3C ) . The activation of ERK1/2 in cytoplasm was observed as usual ( Fig 3A ) . Since with ESAT-6 alone there was no pERK1/2 in the nucleus , but with Na3VO4 treatment there was phosphorylaTION of ERK1/2 , so there might be some putative phosphatases dePHOSphorylating the pERK1/2 as it translocated from cytoplasm to the nucleus . We checked whether sodium orthovanadate alone could induce activation of ERK1/2 ; in the cells stimulated with 1 mM Na3VO4 for the same time points , we found weakly activated ERK1/2 in cytoplasm ( Fig 3E ) and none in the nucleus ( Fig 3G ) . The graphs showing densitomteric analysis of the above ERK blots are shown in Figure 4 . The plots for cytoplasm and nucleus are shown separately . To further confirm the observations from western blotting , kinase assay for ERK1/2 was done . The RAW264.7 cells were treated with LPS and /or ESAT-6 and ESAT-6 and /or Na3VO4 for 60 minutes and the kinase activity was assayed as described in Methods . In cytoplasm ( Fig 5A ) both LPS and ESAT-6 increased ERK enzyme activity over basal level . ESAT-6 treatment was found to have no effect on the ERK kinase activity in the nucleus over the basal level ( Fig 5B ) ; furthermore , ESAT-6 antagonized the LPS -induced ERK activation . Concurrent treatment with Na3VO4 and ESAT-6 increased ERK activation in the nucleus by more than 4-fold compared to the ESAT-6 alone ( Fig 5D ) . Na3VO4 alone did not have any effect on ERK kinase activity over the basal level . Thus the kinase assay confirmed the earlier western blot observations .