Ppagfp n1 vector

Construct myocilin_WT tagged with PAGFP (Addgene, Cambridge, MA) was made by insertion of NheI+BamHI digested myocilin_WT gene (full length, 1–504 amino acids) into NheI+BamHI linearized pPAGFP-N1 vector (Addgene, Cambridge, MA) and transfected into human TM cells. The transfected cells were observed on Zeiss Cell Observer SD (spinning disk) system 24 h post transfection. Myocilin_WT-PAGFP was irradiated by 405-nm light and excited by 488-nm light. Live-cell videos were recorded up to 24 h. Fluorescence of the excited cells at different time points was analyzed by Image J software to quantify the turnover time of myocilin-PAGFP fusion protein. At least 20 cells that displayed low-intensity green fluorescence were analyzed. The relative intensities of fluorescence were plotted with the time and the half-life of myocilin was estimated.

The cDNA sequences encoding ATP5B or DRP1 were amplified by PCR using the oligonucleotide primers (Genomics, Hsinchu, Taiwan). The amplified fragments were cloned into a pPAGFP-N1 vector (RRID: Addgene_11909) or pCMV-HA-N vector (Clontech Cat# 635690), respectively. MOM-GFP plasmid containing mitochondrial targeting sequence of Tom70 fused to pEGFP-N1 vector was a gift from Josef Kittler (RRID: Addgene_127633)^{95 (link)}. Mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase was amplified from DsRed2-Mito-7, a gift from Michael Davidson (RRID: Addgene_55838) by PCR, and cloned into pEGFP-N1 vector backbone to be Mito-GFP plasmid. All clones were verified by sequencing (Genomics). The Escherichia coli DH5α strain was used as the competent cell for the transformation of these constructs. The QIAGEN Plasmid Midi Kit (QIAGEN Cat#12143) was used for the purification of plasmids, and the concentration of the purified plasmids was determined using the NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE). Plasmids were transfected into cells using jetPRIME (Polyplus-transfection Cat#114-15) according to the instructions provided by the manufacturer, and the transfected cells were incubated for at least 24 h before further experiments.