Mcherry gfp lc3

Transfection was achieved using Lipofectamine 2000 Transfection Reagent (Invitrogen, 11668-019) following the manufacturer’s protocol. Cells were transfected with plasmids encoding mCherry-GFP-LC3 (22418) and GFP-LC3 (22405) from Addgene, Cambridge, MA, USA. Human pcDNA3.1(+)-NDRG1 plasmid constructed by Gene-Chem Co. Ltd (Shanghai, China). Human NDRG1 siRNA, HIF-1α siRNA and nontarget siRNA were purchased from Ribobio (Guangzhou, China). Cells were cultured in six-well plates and transfected with plasmids for 24 h. After the designated treatments, live cell images were obtained using a fluorescence microscope (Leica, Wetzlar, Germany). Protein knockdown or overexpression efficiency was assessed by immunoblotting.

The minigene splicing reporter was constructed as previously reported. [35 (link)] Generally, exon 6, intron 6, exon 7 of Tamm41 with flanking genomic sequences were inserted into the vector. Vectors carrying mutant alleles were generated by a PCR-mediated mutagenesis strategy. PINK1-V5 (addgene#13320) and mt-Keima (addgene#56018) was obtained from Addgene. LC3 in vector FUW mCherry-GFP-LC3 (addgene, #110060) was replaced by mitochondrial targeting sequence of the outer mitochondrial membrane protein FIS1 for generating mito-QC. The so got mCherry-GFP-FIS1 was PCR amplified and cloned into pCS2 vector for in vitro transcription. Opa3-pink1 was cloned as previously described [30 (link)]. Zebrafish tamm41, pink1, park2, dnm1l were amplified from zebrafish cDNA. Tamm41-D121A was generated by PCR-mediated mutagenesis strategy from wild type zebrafish tamm41. Human TAMM41 CDS were amplified from AC16 cDNA. For mRNA overexpression experiments, above suggested plasmids were then transcribed in vitro and 2 nl were injected at one-cell stage embryos. Tol2-plasmid was cloned by insertion of zebrafish tamm41 CDS under myl7 promoter (1.6k) as previously reported [54 (link)]. Transgenes were transiently expressed by co-injecting 20 pg of Tol2-plasmid as described above and 80 pg of Tol2 transpose mRNA at one-cell stage.