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. 2013 Mar 20;2(3):415–430. doi: 10.1002/mbo3.83

Figure 6.

Figure 6

Physical interaction between Lsm4 and prions. (A) Colocalization of NM-GFP and Lsm4-mRFP. [PSI+] strain NPK294 was cotransformed with pRS424CUP1p-LSM4-mRFP and pRS415GAL1p-NM-GFP. Transformants were selected on SC-Leu-Trp plate, cultured in SGalSuc-Leu-Trp to mid-log phase to express NM-GFP, further cultured with 50 μmol/L CuSO4 for 3 h to express Lsm4-mRFP, and subjected to confocal microscopy (using Nikon A1) to visualize the two proteins’ intracellular localization. (B) Coimmunoprecipitation of HA-Sup35 and Lsm4-3FLAG. [PSI+] strain NA124 whose genomic SUP35 ORF is replaced by HA-SUP35 was transformed with pRS425GAL1p-LSM4-3FLAG. Transformants were selected on SC-Leu plate, precultured in SSuc-Leu liquid overnight, diluted and cultured in SGalSuc-Leu liquid to induce LSM4-3FLAG expression for 8 h, and subjected to the immunoprecipitation experiment as described in Experimental Procedures. (C) Colocalization of Ure2N-GFP and Lsm4-mRFP. [URE3] strain NPK302 was cotransformed with pRS424CUP1p-LSM4-mRFP and pVTG12 (URE2N-GFP placed under the authentic URE2 promoter; LEU2 marker; single copy). Transformants were selected on SC-Leu-Trp plate, precultured in SC-Leu-Trp liquid overnight, cultured in SC-Leu-Trp with 50 μmol/L CuSO4 to express LSM4-mRFP for 3 h, and subjected to confocal microscopy using Nikon A1.