Different combinations of Agrobacterium GV3101 containing cYFP-CRN78, NbPIP1;1-nYFP, NbPIP2;2-nYFP, or GmPIP2-13-nYFP plasmids were injected into leaves of N. benthamiana; YFP signals were observed under a confocal laser-scanning microscope (Zeiss LSM700, Germany).
For Co-IP assay, indicated plasmid combinations were transiently expressed in N. benthamiana following the methods described above. α-GFP and α-Flag IPs were carried out as per previously described methods [55 (link)]. Total proteins were extracted at 48 hours post infiltration following the methods described above and then incubated with α-GFP (ChromoTek GmbH) or α-Flag beads (Abmart Inc.) overnight. The beads were collected by centrifuge and then washed five times with cold 1xTBS (containing 0.5% Triton X-100). Proteins were released from the beads by incubating at 100°C for 8 min with 50 μL 1xTBS. Immune precipitates were separated by SDS–PAGE gels and detected by immunoblotting with monoclonal α-GFP and α-Flag antibodies (Abmart Inc.).
For Co-IP assay, indicated plasmid combinations were transiently expressed in N. benthamiana following the methods described above. α-GFP and α-Flag IPs were carried out as per previously described methods [55 (link)]. Total proteins were extracted at 48 hours post infiltration following the methods described above and then incubated with α-GFP (ChromoTek GmbH) or α-Flag beads (Abmart Inc.) overnight. The beads were collected by centrifuge and then washed five times with cold 1xTBS (containing 0.5% Triton X-100). Proteins were released from the beads by incubating at 100°C for 8 min with 50 μL 1xTBS. Immune precipitates were separated by SDS–PAGE gels and detected by immunoblotting with monoclonal α-GFP and α-Flag antibodies (Abmart Inc.).