Wipi2

For western blot analysis, cells were harvested in 70 μL lysis buffer (3% SDS, 30 mM Tris base, pH 8.8, 5 mM EDTA, 30 mM NaF, 10% glycerol, 1 mM DTT) and 25–50 μg protein with 6x Laemmli buffer was loaded onto 4–20% Bio-Rad Mini- PROTEAN Precast Gels or 12% self-casted acrylamide/bisacrylamide (29:1) gels. After electrophoresis, the proteins were transferred to PVDF membranes with Bio-Rad Tris/Glycine buffer or self-made transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3) and stained with the following antibodies: ubiquitin – FK2 clone Millipore, 04–263; mCherry – Novus Biologicals, NBP1-96,752; GFP – Santa Cruz Biotechnology, sc-9996; LC3B – Cell Signaling Technology, 2775; SQSTM1/p62 – Sigma-Aldrich, P0067; ZFTVE1/DFCP1 – Cell Signaling Technology; WIPI2 – Invitrogen; ACTN2 (actinin alpha 2) – Sigma-Aldrich, A2543 or A7811; RPS6 (ribosomal protein S6) – Cell Signaling Technology, 2771; GAPDH – HyTest, 5G4. Western blots were either detected with LI-COR Biosciences Odyssey (Figures 4A, E, G, M and 5D) or Biorad ChemiDoc (Figures 4C, I, J, K and 5E).

After fixation, cells were stained for the cardiomyocyte marker TNNI3 (troponin I, cardiac 3; Millipore, MAB1691), nuclei (DAPI or DRAQ5; Thermo Fisher Scientific, D1306) and antibodies against the protein of interest (LC3B – Cell Signaling Technology, 2775; ZNF418 – Sigma-Aldrich, SAB2700695; FLAG – Sigma-Aldrich, F3165; ZFYVE1/DFCP1 – Cell Signaling Technology, 85156S; WIPI2 – Invitrogen, PAS-54,098). Slides were imaged using a Nikon Eclipse Ti inverted microscope with 10–20x air objective, Nikon A1R confocal microscope with 20–60x objective or a Zeiss Axiovert microscope with 40x or 63x oil objective and 7 images/well were captured. Images were analyzed with NIS Elements software.