Cat m20004

To determine the subcellular localization of Flot1 and RinRK1 in N. benthamiana leaves, A. tumefaciens EHA105 cells were used for the agroinfiltration of N. benthamiana leaves with constructs along with p19. After 2–3 days, the leaves were examined using a confocal microscope (TCS SP8, Leica). The excitation/emission filter sets for GFP were 488 nm/498–540 nm. We used an anti-GFP antibody (Abmart, CAT.M20004) for the immunoblot analysis.
To determine the subcellular localization of RinRK1, Flot1, NFR1, and NFR5 in L. japonicus hairy roots, A. rhizogenes AR1193 cells carrying the indicated constructs were used to transform L. japonicus hairy roots. The transgenic roots were then inoculated with M. loti R7A/mTag, M. loti R7A/NF, or M. loti nodC. The subcellular localization of the proteins was determined using a spinning disk confocal microscope (REVOLUTION XD, ANDOR). The excitation/emission filter sets were as follows: GFP (488 nm/498–540 nm), mCherry/DsRed (561 nm/570–610 nm), and mTag (405 nm/415–454 nm). The proportion of aggregated proteins at the root-hair tip was analyzed in the infection zone at the same magnification. The protein subcellular localization assays were repeated at least twice, more than 100 root hairs from six composite plants were analyzed by live-cell imaging each time.

Different combinations of constructs (NFR1-Myc, NFR5-Myc, MtLYK10-Myc, RinRK1-Flag, RinRK1-N-Flag, and Flot1-eGFP) were inserted into N. benthamiana leaves via agroinfiltration along with p19. The plants were incubated for 48 h in a growth room with a 16-h light (250 µmol/m²/s)/8-h dark cycle. The N. benthamiana leaves were ground in liquid nitrogen. The ground material was resuspended in extraction buffer (50 mM Tris-MES, pH 8.0, 0.5 M sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT, 1 mM PMSF, and Complete Mini protease inhibitor cocktail tablets). The extracts were centrifuged at 13,400 × g for 15 min at 4 °C and the supernatants were collected for the Co-IP assays. We used GFP beads (SMART) and Flag beads (HuiOu Biotech) for the immunoprecipitation. For the immunoblot analysis, proteins were separated in 6%, 8%, or 10% SDS-PAGE gels and electroblotted to a pretreated polyvinylidene fluoride membrane at 120 V for 90 min. The membrane was blocked using TBS buffer containing 5% skimmed milk powder for 2 h at room temperature. The membrane was then sequentially incubated with the primary antibody at 4 °C overnight and then with the secondary antibody diluted in TBS buffer containing 1% skim milk powder for 1 h at room temperature. The anti-GFP (Abmart, CAT.M20004), anti-Myc (Abmart, CAT.M2000M), and anti-Flag (HuiOu Biotech, CAT.HOA012FL01) antibodies were used.