Despite important progress in recent decades, mortality remains high among patients with HF

otGABARAPL2 were derived by PCR-based transfer of human GIMAP6 and GABARAPL2 from full-length cDNA clones BC074744 and BM544477, respectively, into the EcoRI-XhoI site of pcDNA3Biot1His6iresBirA. Plasmids encoding N-terminally myc-tagged GIMAP proteins or N-terminally HA-tagged proteins were derived by cloning the corresponding cDNA sequences into plasmids pCANmyc1, pCANmyc3 or pCANHA1, respectively. c) Protein expression vectors for stable cell lines. Plasmid pmycBirA-ires-neo was derived by insertion of a PCR-derived myc-tagged BirA fragment between the BamHIEcoRI sites in plasmid pIRES-neo. Plasmid biotGIMAP6-His-pCAG-iPuro was derived by transfer of a PCRderived DNA fragment spanning the biotinylation tagGIMAP6-6His sequence in plasmid biotGIMAP6 into the SalI site of plasmid pCAGiPuro. Materials and Methods Materials Antibodies were sourced from the following companies: antiMAP1LC3A, anti-MAP1LC3B, anti-GABARAP, anti-GABARAPL1 anti-SQSTM1, and anti–ACTIN were 23178882 from Sigma-Aldrich; anti MAP1LC3C was from Abcam; anti-CYCLIN D1 was from Calbiochem. Rat monoclonal antibodies to both human GIMAP6 and GABARAPL2 were generated in-house. A rabbit polyclonal antiserum to human GIMAP6 was produced by Harlan Laboratories to an in-house generated antigen. Inhibitors were from the following sources: PP242 was from Cambridge Bioscience UK; emetine and chloroquine were from Sigma-Aldrich; AZD8055 was a gift from Dr Sylvie Guichard, Astrazeneca UK. G418 and penicillin/streptomycin were from Invitrogen; all other selective antibiotics used in mammalian cell culture were purchased from InvivoGen. Cell Line purchase c-Met inhibitor 2 Establishment and Maintenance a) Myc-BirA-Jurkat cells. Jurkat T cells were routinely maintained in RPMI/10% fetal calf serum/100 units/ml penicillin/100 g/ml streptomycin. For establishment of myc-BirA-Jurkat cells, approximately 107 Jurkat T cells were transfected by electroporation in RPMI with 20 g of MluI-linearized pmycBirA-iresneo. Cells were allowed to recover overnight in complete medium. The following day, the cells were spun down and resuspended in 20 ml of complete medium and plated at 100 l/well into 96-well plates. The next day an equal volume of complete medium supplemented with 1 mg/ml G418 was added to each well. Every three days thereafter, half of the medium was replaced with fresh complete medium containing 500 g/ml G418. myc- 2 GIMAP6 Interacts with GABARAPL2 BirA expressing cell lines were identified by Western blotting. A single myc-BirA-Jurkat cell line was selected for subsequent use. b) Biot-GIMAP6-His myc-BirA Jurkat cell line. The mycBirA-Jurkat cell line was maintained in complete medium containing 9761423 500g/ml G418. Approximately 107 cells were transfected by electroporation with 20 g of either PvuIlinearized plasmid biot-GIMAP6-His-pCAG-iPuro or the corresponding vector. Cells were allowed to recover overnight in complete medium containing 500 g/ml G418. The following day, the cells were spun down and resuspended in 20 ml of complete medium containing 500 g/ml G418 and plated at 100 l/well into 96-well plates. The next day an equal volume of complete medium containing 500 g/ml G418 and 6 g/ml puromycin was added to each well. Every three days thereafter, half of the medium was replaced with fresh complete medium containing 500 g/ml G418 and 3 g/ml puromycin. Cells carrying the parental pCAG-iPuro vector were isolated in parallel, as controls. Biot-GIMAP6-His expressing clones were identified by Western blotting of cell lys