Pact2 ad vector

The COOH-terminus of TRPM7 (residues 1120–1862) was subcloned into pBMT116 LexA vector to make the “bait” vector LexA-CTERM. A rat brain prey library in the pACT2 AD vector was purchased from Clontech Laboratories, Inc (Mountainview, CA). A yeast-two-hybrid screen was conducted following the manufacturer’s protocol for large-scale transformation. Positive interactions were identified by growth on selective media and a beta galactosidase assay before sequencing of purified plasmids. To conduct the directed Y2H beta galactosidase assay, the coiled-coil (CC) domain (residues 1120-1288) and kinase (KIN) domain (residues 1597–1862) of TRPM7 were subcloned into pBMT116 to make LexA-CC and LexA-KIN. The kinase-inactive LexA-CTERM (K1646R) mutant was made using QuikChange (Agilent Technologies, Santa Clara, CA) following the manufacturer’s protocol. The primers used to make the Y2H “bait” vectors are described in Supplementary Methods.

The full length CDS Drosophila Imp and Lin28 cloned in pACT2 AD vector (Clontech) to produce Gal4 transcriptional activation domain (TAD) and LexA DNA binding domain-(DBD) to generate TAD-Lin28 and TAD-Imp; and DBD-Lin28 and DBD-Imp respectively [42 (link)]). All constructs were verified by DNA sequencing. Various combinations of plasmids expressing TAD and DBD were transformed into the yeast strain YPH500 (MAT α, ade 2, his3, leu 2, lys2, trp1, ura3) harboring the pSH18-34 plasmid (lexAop- LacZ reporter) by the standard lithium acetate method. Independent transformants for each combination were patched onto glucose plates containing X-gal for 24–48 hours, followed by a beta-galactosidase liquid assay as described previously [43 (link)].