Cloning of expression constructs

Ectopic Myc-tagged PP2A Aα subunit was expressed from pLPC-N MYC (Addgene, plasmid # 12540). For the LRKRGST sequence context, the PP2A Aα cDNA was amplified by reverse transcription PCR from HeLa cells and inserted via the Bam HI/Xho I sites. For the NGST sequence context, the complete Myc–PP2A Aα cassette was excised from a different plasmid (pBABE-Puro-Aα) by Hind III/Xho I and inserted into pLPC-N MYC via the Hind III/Xho I sites. Codon-optimized cDNAs of the rat Myc-PP2A B55α fragments corresponding to six different Myc tag sequence contexts [NGST, IGST, IAST, LRKR, TRKR, and L(GS)₃; Table 1], three different 6xMyc tag sequence contexts (fig. S2), or four different Myc tag sequence contexts (designated as pCMV-Myc-N, pCMV-Myc-C, pCruz-Myc, and pcDNA3.1-Myc B; Table 2) were synthesized and cloned into the pET23a(+) bacterial expression vector via the Nde I/Xho I restriction sites by GenScript Inc. All four latter versions contain an in-frame stop codon immediately upstream of the Xho I restriction site to avoid read through into the 6xHis tag sequence of the vector. The exact DNA sequences can be obtained upon request.