Telaprevir VX-950 and the remaining sites SQ Artemis. W While DNA PK efficiently phosphorylates all mutants S4A Artemis, ATM was not effectively phosphorylate Artemis that either. S503A, S516A or S645A mutation, the specificity of t these sites for ATM and interdependence recognizable for the phosphorylation of these sites DNA PK, but independent Ngig multiple locations in Artemis. Of 10 S / TQ site Artemis, eight in the C-terminal H Half of the protein. Examination of the N and C-terminal fragments and Volll Nts protein mutates all m Shown resembled C terminal phosphorylation as substrates for DNA-PK or ATM and Artemis that phosphorylation was exclusively Lich within the end of C was at specific sites. Anything similar data with wild-type insects and 9A Artemis expresses found.
Interestingly, w While this manuscript was in the year Soubeyrand et al identified six sites of DNA PK phosphorylation in Artemis is not in line with our results, but unlike SQ sites previously by Ma et al. As Soubeyrand et al also physiologically relevant ionic conditions phosphorylated Artemis, the controversy over the identity of t Phosphorylation of Artemis preparation is probably explained by technical differences in salt concentration Explained in more detail. To examine the phosphorylation in vivo, we generated a phospho-antique Body against Artemis S645. aArtemis pS645 was immunoblotted against GSTArtemis not either S645A or S562A mutations and WT specifically detected S645A and S562A but Artemis Artemis.
To investigate Artemis S645 phosphorylation in vivo, WT, ATM-deficient and Artemis-deficient fibroblasts with 0, 10 or 20 Gy IR and 15 were irradiated min after irradiation, cell extracts were immunpr Zipitiert and Artemis pS645 aArtemis immunoblotted and aArtemis. IR induces a mobility shift h Depends ATM Artemis. aArtemis pS645 selectively detects a signal from irradiated and non-irradiated cells from WT cells or non-irradiated cells or SCID AT RS. Gr Ere specificity Was t best by phosphatase treatment and the addition of phospho-peptide competition CONFIRMS. Remarkably, WT cells showed normal with DNA PK inhibitor NU7441 treated specific kinase induction aArtemis pS645 after IR, suggesting that Artemis phosphorylation in vivo is not dependent Ngig of DNA-PK activity t.
aArtemis pS645 was unspecific immunofluorescence and immunoblotting without prior Immunpr zipitation Artemis. We conclude that S645 Artemis is a phosphorylation in vivo ATM. Mutation of the phosphorylation sites of DNA KP / ATM in Artemis t had no effect on the activity Artemis in vitro or in vivo After checking the Artemis phosphorylation in vitro and in vivo, we followed its functional implications. To verify that Artemis 9A includes main sites of phosphorylation in vivo, we examined the IR-induced hyperphosphorylation in vivo. After transient transfection of cDNA 9A Artemis is the mobility T 9A Artemis unaffected by irradiation, unlike WT Artemis. Thus occurs IR induced hyperphosphorylation Artemis at one or more of the identified sites. We have coins then examined whether WT and 9A Artemis was the erg already desc .