Construction and Validation of Fluorescent Protein Fusion Constructs

The PMP34-GFP^{66 (link)} and PMP34-GFP-UBko^{19 (link)} constructs used in this study were previously generated using standard protocols, where the PCR product of the ORF of PMP34 was ligated into the EcoRI and BamHI site of the pmGFP-N1 vector (Clontech). Ub-KO (G76V), a ubiquitin where all seven lysines were mutated to arginines, was excised from GFP-Ub-KO (G76V) (Addgene 11932) with BsrGI and NotI, and the resulting fragments were ligated into similar sites in PMP34-GFP. The Cherry-LC3 construct was previously generated²⁷ , where the PCR product of the LC3-B ORF was ligated into the BglII-EcoRI site of the mCherry-C1 vector (Clontech) using standard protocols. LAMP1A-mEmerald was donated by Dr. Sergio Grinstein (Hospital for Sick Children, Toronto, ON, Canada), and FLAG-ULK1-wild type was donated by Dr. John Brumell (Hospital for Sick Children, Toronto, ON, Canada). The siRNAs used in this study were custom synthesized from Sigma-Aldrich and are listed in Supplementary Table 2. siRNA knockdown was validated by immunoblot or TaqMan real-time quantitative PCR.