1. Oligomerization of PCR2
Oligomerization of PCR2.(A) Membrane proteins isolated from ZHY3 (zrt1 zrt2) yeast cells transformed with a V5-tagged pYES2 NT/C vector containing Arabidopsis PCR2 (NCT-PCR2).
Oligomerization of PCR2.(A) Membrane proteins isolated from ZHY3 (zrt1 zrt2) yeast cells transformed with a V5-tagged pYES2 NT/C vector containing Arabidopsis PCR2 (NCT-PCR2). Cross-linking was performed using 0, 1, 3, and 5 mM EGS, and the PCR2 protein was detected by immunoblotting with an anti-V5 antibody. In addition to the monomer being detected at 19 kD (single dot), a band corresponding to a putative dimer was detected at 37 kD (double dots). Higher EGS concentrations resulted in high molecular mass complexes.(B) Membrane proteins of yeast cells expressing the vector containing PCR2 with a V5 tag at the C terminus (CT-PCR2), the vector carrying a V5 tag at the C terminus and a His tag at the N terminus (NCT-PCR2), or both (CT-PCR2 and NCT-PCR2) at the same time (last lane). Cells were grown in SG-ura-leu medium to induce gene expression. Subsequently, proteins were extracted using a buffer with (+DTT) or without DTT (−DTT), separated by SDS electrophoresis without prior treatment with a cross-linker, and detected by immunoblotting using V5 antibodies. The arrow in the last lane indicates a putative dimer (36 kD) consisting of one molecule of NCT-PCR2 (19 kD) and one molecule of CT-PCR2 (17 kD). Dots indicate the oligomers containing NCT-AtPCR2, while asterisks indicate oligomers containing CT-PCR2.
Won-Yong Song et al. Plant Cell 2010;22:
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