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Briefly, yeast mutants were grown in YPD medium containing 2% galactose and 100 mM UPI. Western blotting and activity assay of wild type and each mutant purified from yeast were performed as described above. Yeast two-hybrid assay The yeast two-hybrid assay was performed using the manufacturer’s protocol (Clontech, CA, USA). The full-length C-terminus of Nrx1 (N-terminal 63 amino acid residues) was inserted into pB27 vector. The full-length C-terminus of Camk2a (N-terminal 349 amino acid residues) was inserted into pPC86 vector. The full-length N-terminal sequence of LePrc1 was inserted into pEG202 vector. The full-length N-terminal sequence of Ire1 (N-terminal 496 amino acid residues) was inserted into pJG4-5 vector. To analyze Nrx1-Camk2a, Nrx1-LePrc1, or Nrx1-Ire1 interaction, transformed yeast cells were grown in SD-LEU-TRP medium (0.17% yeast nitrogen base, 0.5% yeast extract, and 2% raffinose) with 2% galactose as a carbon source and without leucine or tryptophan. To analyze interaction between Camk2a and LePrc1, Camk2a-Myc was inserted into pJG4-5 and LePrc1-HA was inserted into pPC86. To analyze interaction between Camk2a and Ire1, Camk2a-Myc and Ire1-HA were inserted into pJG4-5 and pPC86, respectively. To analyze interaction between LePrc1 and Ire1, LePrc1-Myc was inserted into pPC86 and Ire1-HA was inserted into pEG202. To analyze interaction between Nrx1 and LePrc1, Nrx1-Myc was inserted into pPC86 and LePrc1-HA was inserted into pEG202. All constructs were transformed into the AH109 yeast strain. Western blotting Yeast cells were grown in YPD medium and collected by centrifugation at 2,000 × g for 5 min. The cells were resuspended in ice-cold lysis buffer (100 mM Tris-HCl, pH 7 847798691e