Nh2 pprkprrwrn s po3h2 is cooh

For yeast two-hybrid analyses, the individual constructs were cloned into the vectors pGADT7 and pGBKT7 and co-transformed into the yeast strain PJ69-4A. Activity of the ADE2 reporter was analyzed by growth of co-transformed yeast on SD medium lacking adenine. SDS-PAGE, Western blotting and immunodetection followed standard procedures. Total proteins were extracted from 3-day-old etiolated Arabidopsis seedlings (50 seedlings) or transiently transformed N. benthamiana leaves (2 leaf disks) by directly grinding in 100 µl 2 x SDS sample buffer under red safe light illumination. Chemiluminescence detection was performed with an Amersham Image Quant800 (Cytiva) system.
The rabbit anti-NPH3-S744P antibody was generated with the phosphorylated synthetic peptide NH2-PPRKPRRWRN-S(PO3H2)-IS-COOH followed by affinity-purifications against the non-phosphorylated and phosphorylated peptide at Eurogentec (Liege, Belgium).

For yeast two-hybrid analyses, the individual constructs were cloned into the vectors pGADT7 and pGBKT7 and co-transformed into the yeast strain PJ69-4A. Activity of the ADE2 reporter was analyzed by growth of co-transformed yeast on SD medium lacking adenine. SDS-PAGE, Western blotting and immunodetection followed standard procedures. Total proteins were extracted from 3-day-old etiolated Arabidopsis seedlings (50 seedlings) or transiently transformed N. benthamiana leaves (2 leaf disks) by directly grinding in 100 µl 2 x SDS sample buffer under red safe light illumination. Chemiluminescence detection was performed with an Amersham Image Quant800 (Cytiva) system.
The rabbit anti-NPH3-S744P antibody was generated with the phosphorylated synthetic peptide NH2-PPRKPRRWRN-S(PO3H2)-IS-COOH followed by affinity-purifications against the non-phosphorylated and phosphorylated peptide at Eurogentec (Liege, Belgium).