Figure 1

Western blot analyses of rice NH and TGA proteins fused to YFPN or YFPC and expressed in protoplasts. (A) and (B): Constructs encoding YFPN (YN)-NH1, −NH2, −NH3, −NH4, and –NH5, or YFPC (YC)-NH1, −NH2, −NH3, −NH4, and –NH5 were used to transfect rice protoplasts. A Ubi-Gus plasmid was included as an internal control for transfection. Protoplast cells were harvest 24 hours after transfection. Each protein sample was suspended in 1× SDS loading buffer and amount of loading was adjusted based on GUS activity. The negative control contained plasmids pSY736, carrying YN, and pSY735, carrying YC. (A) Western blot analyses were carried out using an α-Myc or an α-HA tag monoclonal antibody. (B) Transfections of protoplasts were carried out individually with an YN-NH plasmid together with an YC-TGAL1 plasmid, or an YC-NH plasmid with an YN-TGAL1 plasmid. The negative control sample was the same as above. The nitrocellulose membranes were probed with the α-Myc and α-HA antibodies simultaneously. YC-fused TGA2.1, TGA2.2, TGA2.3, rLG2, TGAL1, TGAL2, TGAL4, TGAL5, TGAL6, TGAL7, TGAL8, TGAL9, and TGAL11 were expressed in protoplasts alone in (C), or with YN-NH1 in (D), YN-NH2 in (E), or YN-NH4 in (F). YC fusions were probed with α-HA and YN-fusions probed with α-Myc antibodies. Molecular weight: YFPN = 22.0kD; YFPC = 14.5kD; NH1 = 63.9kD; NH2 = 67.4kD; NH3 = 65.0kD; NH4 = 53.3kD; NH5 = 51.8kD; TGA2.1 = 37.2kD; TGA2.2 = 37.1kD; TGA2.3 = 36.8kD; rLG2 = 55.7kD; TGAL1 = 51.3kD; TGAL2 = 36.9kD; TGAL4 = 52.9kD; TGAL5 = 48.7kD; TGAL6 = 40.6kD; TGAL7 = 51.3kD; TGAL8 = 49.3kD; TGAL9 = 48.1kD; and TGAL11 = 52.9kD. Each protein is marked with a white arrowhead. The black “*” symbol indicates nonspecific bands detected by the α-Myc antibody.