Fig. 2: Physical interaction of GAPC with NF-YC10.
a Expression and purification of recombinant NF-YC10 produced in E. coli. Proteins from pellet (P) or supernatant (S) following centrifugation of E. coli cell lysate were separated, and NF-YC10-6xHis was probed by immunoblotting (IB) with an anti-6xHis antibody (left panel). Protein marker size is on the left. NF-YC10 was nickel affinity-purified from the E. coli and shown here on polyacrylamide gel stained with Coomassie blue (right panel). M, marker. b Co-immunoprecipitation of GAPC and NF-YC10. GAPC1-Flag, GAPC2-Flag, or empty vector control (EV) was mixed with NF-YC10-6xHis. Immunoprecipitation (IP) was performed using an anti-Flag antibody, then immunoblotting (IB) was performed using the antibodies indicated on the right. NF-YC10 protein input is shown on polyacrylamide gel stained with Coomassie blue (bottom panel). c Bimolecular fluorescence complementation (BiFC) analysis. GAPC-YFPN and NF-YC10-YFPC were transiently co-expressed in tobacco leaves and observed under a confocal microscope. GPA1 and PLDα1 that bound each other were used as controls. Scale bars = 10 µm. d Quantification of BiFC. Fluorescence intensity of tobacco leaves used in c was measured by ImageJ software. Values are average ± S.D. from 10 randomly chosen regions of tobacco leaves infiltrated. Black dots represent individual data points.
