Co-Immunoprecipitation of Myc and LPA1-GFP

IDD3-Myc + LPA1-GFP, IDD13-Myc + LPA1-GFP, IDD10-Myc + LPA1-GFP, IDD3-HA + IDD13-Myc, or IDD3-HA + IDD13-Myc + LPA1-GFP were coexpressed in N. benthamiana leaves, respectively. After 36 h of expression, the protein was extracted, and Co-IP assays were performed as described previously (Kim et al. 2009b (link)). Twenty micrograms of protein from each sample were separated on a 10% SDS-PAGE gel and electrotransferred onto Immobilon-P Transfer Membranes (MILLIPORE JAPAN, Tokyo, Japan). For the subsequent western blot analysis, the following primary antibodies were used: an anti-HA antibody (1:2000; Abcam, Cambridge, MA, USA), anti-GFP antibody (1:2000; Abcam), and anti-Myc antibody (1:2000; Abcam). The membranes were incubated for an additional hour with an anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA) before the signal was detected using an ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).