Pbifc vc155

To visualize protein binding, plasmids were created for bimolecular fluorescent complementation (BiFC) of Venus using N-terminal (VN) and C-terminal (VC) reporter fragments as previously described (Kodama and Hu, 2010 (link)). In brief, the coding region of each cDNA (MCUR1, MCU, MICU1, EMRE and PPIF) was amplified by PCR and cloned into pBiFC-VC155 (addgene Plasmid 22011) to generate the corresponding cDNA-VC protein expressed in frame. Similarly, all the cDNAs were also cloned into pBiFC-VN155 (addgene Plasmid 27097) to generate the corresponding cDNA-VN protein expressed in frame. HeLa cells were plated in six-well plates containing 0.2% gelatin- coated glass coverslips and transfected with corresponding plasmid encoding for MCU complex component proteins fused with -VN and -VC reporter fragments. After 36 hours, cells were loaded with Tetramethylrhodamine, methyl ester (TMRM; 100 nM) for 30 min at 37°C for mitochondrial staining. Note: Basal autofluorescence was observed in non-expressing cells that was considered negative. Image acquisition was performed using a Carl Zeiss 510 confocal microscope using a 63× oil objective with excitation at 514 and 561 nm respectively. Intensity profile were created using line scan for relative fluorescence of BiFC/TMRE using Carl Zeiss ZEN 2010 Imaging Software.