pMXs-puro-GFP-WIPI1 and pMXs-puro-GFP-DFCP1 were a kind gift from Dr. N. Mizushima (University of Toyko, Japan) and pMXs-IP-GFP-ULK1 was purchased from Addgene (#38193). To generate pBMN-mEGFP-C1, mEGFP-C1 (Addgene #36412) was PCR amplified (together with the multiple cloning site) and cloned into pBMN-Z at BamHI/SalI sites using the Gibson Cloning kit (New England BioLabs) according to manufacturer's instructions. The BamHI and SalI sites used to insert mEGFP-C1 were not regenerated. The following GFP-tagged plasmids were generated by PCR amplification of open reading frames followed by ligation into pBMN-mEGFP-C1: OPTN, NDP52, p62, TAX1BP1, NBR1, LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2. The Gateway Cloning (Invitrogen) system was used to generate GFP-, mCherry-, myc- and FLAG/HA-constructs. Briefly, TBK1, TBK1-K38M, NDP52, OPTN, p62, DFCP1, WIPI1 and ULK1 were cloned into pDONR2333. Mutations in cDNA sequences were introduced using PCR site directed mutagenesis in the pDONR2333 vector, (sequences of mutagenesis primers used are available upon request) then recombined into pHAGE-N-FLAG/HA, pHAGE-N-GFP, pHAGE-N-mCherry and/or pDEST-N-myc using LR Clonase (Invitrogen) as per the manufacturer's protocol. All constructs generated in this study were verified by sequencing.
To generate stably transfected cell lines, retroviruses (for pBMN-mEGFP-C1 constructs, pBMN-mCherry-Parkin, pBMN-puro-P2A-FRB-Fis1, pCHAC-mt-mKeima-IRES-MCS2) and lentiviruses (for pHAGE- and pDEST- constructs) were packaged in HEK293T cells. HeLa cells were transduced with virus for 24 h with 8 μg/ml polybrene (Sigma) then optimized for protein expression via selection (puromycin or blasticidin) or fluorescence sorting.