Complete protease inhibitors 1x

CoIP assays were performed on N. benthamiana agro-infiltrated leaves with p35S::IDD2-RFP, p35S::TCP14-RFP, p35S::RGA-GFP or p35S::mRGA-GFP. Three days after infiltration, total proteins were extracted with the native extraction buffer [Tris-HCl (pH 7.5) 50 mM, glycerol 10%, nonidet P-40 0.1% supplemented with Complete Protease Inhibitors 1X (Roche)], and then incubated for 2 h at 4 °C with 50 µL of anti-GFP antibody conjugated with paramagnetic beads (Miltenyi Biotec, Catalog number 130-091-125). After incubation, samples were loaded onto a magnetic column system (µ columns; Miltenyi Biotec) to recover the immunoprotein complexes. The immunoprecipitated (RGA-GFP and mRGA-GFP) and co-immunoprecipitated (IDD2-RFP and TCP14-RFP) proteins were detected by western-blot with a 2000-fold dilution of anti-GFP (JL8; Clontech, Catalog number 632380) and anti-RFP (6G6 α-Red, Chromotek, Catalog number 51020014AB), respectively.

Feeder-free ESC and EB were homogenized in lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Igepal, Complete protease inhibitors 1X (Roche), 1 mM NaVO₄ and 10 mM NaF]. Then, 40 μg of total protein was separated on 10% SDS-PAGE at 100 V for 180 min and transferred overnight at 30 V and 4°C to polyvinylidene fluoride membranes (Hybond-P, Amersham Pharmacia, GE Healthcare). Membranes were blocked for 1 h in milk or 5% albumin at room temperature and incubated with primary antibody (Table S3) in blocking solution overnight at 4°C, then three times rinsed and incubated with secondary antibody coupled to a peroxidase HRPT (Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was developed using a luciferase-based reaction (ECL or Immobilon Millipore) and sensitive photographic film. Densitometric analysis was done using ImageJ software, and data were normalized against GAPDH and expressed as relative protein levels versus controls.