Su9 egfp

Tfrc promoter reporter construct was generated by cloning a fragment from −897 bp to +129 bp around mouse TfR1 transcription site into pGL3basic reporter. Tfrc 3’UTR luciferase construct was described previously (Bayeva et al., 2012 (link)). IRE sites in this construct were deleted using QuickChange Site-Directed Mutagenesis Kit (Agilent). pLenti-rtTA3, SU9-eGFP, and pBI-eGFP plasmids were purchased from Addgene. Human ABCB7 cDNA plasmid was a generous gift from Dr. Berry Paw (Brigham and Women’s Hospital and Boston Children’s Hospital). Human MIA40 and ALR cDNA clones were purchased from Open BioSystems. Coding sequence of Mia40, ALR, ABCB8, SU9-eGFP, and ABCB7 were tagged with 6x-His tag and subcloned into pBI-eGFP (Addgene) for import experiment. ABCB8 with 6x-His or 3x-Flag tag in the C terminus and Mia40 with 6x-His tag in the C terminus were inserted into pCMV6-XL6. Cysteine mutants of ABCB8 were generated using PCR-based site-directed mutagenesis. All constructs were sequenced before experiments.

For the generation of the construct MTS-Abeta-GFP, pcDNA5/FRT/TO (Thermo) was used as a backbone. The following inserts were amplified by Q5 High-Fidelity DNA Polymerase (NEB) and cloned into the backbone via NEBuilder HiFi DNA Assembly Master Mix (NEB): MTS (2× COX8 presequence in tandem) amplified from pCMV CEPIA2mt (Addgene, catalogue no. 58218), Abeta (Aβ1-42) amplified from HeLa wild-type complementary DNA (cDNA) with primers (5′-TCC ATG CGG GGT TCT GAT GCA GAA TTC CGA CAT GAC TCA GGA TAT G-3′ and 5′-CTC GCC CTT GCT CAC GGA TCC CGC TAT GAC AAC ACC GCC CAC C-3′, containing a GS linker) and enhanced green fluorescent protein (EGFP) amplified from Su9-EGFP (Addgene, catalogue no. 23214).