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. 2020 Mar 4;11:232. doi: 10.3389/fpls.2020.00232

FIGURE 2.

FIGURE 2

Validation of selected AtNHR2A and AtNHR2B interactions. Protein extracts of N. benthamiana transiently expressing AtNHR2A-GFP were mixed with His-AtENGD-1 purified from E. coli (Rosetta) and subjected to immunoprecipitation with anti-GFP antibodies. Co-immunoprecipitation of His-AtENGD-1 (red asterisk) was detected by Western blot using anti-His antibodies (A). AtNHR2B-GFP and Myc-AtRPN1A pair and AtNHR2B-GFP and HA-AtCCoAMT1 pair were transiently co-expressed in N. benthamiana and immunoprecipitated with anti-Myc and anti-HA antibodies, respectively. Co-immunoprecipitation of AtNHR2B-GFP in each experiment (red asterisks) were detected by Western blot using anti-GFP antibodies (B,C). Expected protein sizes are shown by arrows. AtNHR2B, or its non-functional version AtNHR2B(1–140) fused to the N-terminal fragment of EYFP were co-expressed in N. benthamiana with AtCCoAOMT1 fused to the C- terminal fragment of EYFP. The reconstitution of the EYFP signal was evaluated via laser scanning confocal microscopy at 3 days after infiltration (D). Images were taken using excitation wavelength of 514 nm and an emission wavelength of 500 to 530 nm. Bar = 10 μm.