|- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure
- 25X SAM is not stable, and loses activity within a few hours after
Plasmids and proteins
Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7,8. Recombinant active S6K132 and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33. Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen). Full paper
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