Figure 3
Translocation of AIF from mitochondria to parental macronucleus. A. Map of expression vector, named AKgfpTtAIF, with neomycin resistance cassette (Neor-cassette), 5'UTR and ORF of AIF, codon-optimized GFP (GFP-cassette), replication origin derived from Stylonychia lemnae and telomeres from Tetrahymena. Neomycin resistance is expressed under control of β-tubulin promoter. Before biolistic bombardment, the plasmid was digested with SfiI to expose telomere sequences on both ends. B. After biolistic bombardment, cytoplasmic localization of AIF::GFP was detected using α-GFP. This fluorescent pattern was coincided with MitoTracker Green (MTG). No-bombardment indicates non-transformed wild-type cell. Scale bar in photograph indicates 10 μm. C. Translocation of AIF::GFP was visualized with α-GFP antibodies. White arrows indicate parental macronucleus. Overlay represents fusion image of blue (nuclei) and red (AIF::GFP) fluorescence. Scale bar in photograph indicates 10 μm. D. Fluorescence microscopy of living cells expressing AIF::GFP at the stage of MacIIp. Red arrows indicate AIF::GFP in parental macronuclei. pMa denotes parental macronucleus.