Subcellular localization of PiCYP74C9::GFP protein by cell fractionation and immunoblot analysis. Leaves of a transgenic tobacco plant expressing the PiCYP74C9::GFP fusion protein were fractionated into total leaf extract, chloroplasts, mitochondria, peroxisomes, and tonoplast. Proteins were separated on a 12.5% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with antibodies against GFP, the large subunit of Rubisco (Rubisco-LS), coupling factor 1 of chloroplastic ATPase (Cp-ATPase CF1), mitochondrial ATPase alpha subunit (Mt-ATPase alpha), catalase, vacuolar membrane proton-translocating inorganic pyrophosphatase (V-PPase), and binding protein (BiP). Protein amounts loaded were 5 μg in A or 2.5 μg in B for total leaf extracts, 3 μg for chloroplasts, 0.3 μg for mitochondria and peroxisomes, and 0.15 μg for tonoplast. Sample denaturation with SDS was done at 100°C for 3 min in A and at 50°C for 20 min in B.