α lc3

HEK293T, HeLa, or MEF cells were grown in chamber slides (Thermo Fisher), or on cover slips (Chemglass) in 24-well plates, and transfected and/or infected as indicated. Cells were fixed with 4% (w/v) paraformaldehyde (PFA, Santa Cruz) for 20 min, permeabilized with 0.5% (v/v) Triton-X-100 in PBS, and then blocked with 10% (v/v) goat serum or 1% milk powder in PBS for 1 h. For immunostaining, α-TRIM23 (1:200, ab97291, Abcam), α-LC3 (1:400, Novus Biologicals), α-phospho-S172-TBK1 (1:400, 5483, Cell Signaling), α-TBK1 (1:400, #3013, Cell Signaling), α-TRIM23 (1:100, clone C-1, sc-393923, Santa Cruz), α-FLAG (1:400, M2, Sigma), α-p62 (1:400, PROGEN Biotechnik), α-LAMP1 (1:400, H4A3, Developmental Studies Hybridoma Bank), α-V5 (1:500, R960-25, Life Technologies), α-myc (1:400, 9B11, Cell Signaling) and α-ATG16 (1:200, A7356, Sigma) were used, followed by incubation with secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 633, or Alexa Fluor 647 (all 1:400, Life Technologies). Cells were mounted in DAPI-containing Vectashield (Vector Labs) to co-stain nuclei. All laser scanning images were acquired on an Olympus IX8I confocal microscope or on a Leica SP8 confocal microscope. Cytoplasmic GFP-LC3B puncta in HeLa, HEK293T and MEF cells were manually counted for 30 or 50 randomly selected cells.

75 μg of protein per well were loaded into NuPAGE 12% Bis-Tris gels (Life Technologies, Carlsbad, CA). After electrophoresis and transfer to PVDF membranes Odyssey blocking buffer was applied (Li-Cor, Lincoln, NE) for 1 h at room temperature. Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies diluted in Odyssey blocking buffer including 0.1% Tween-20 and 0.05% NaN₃: α-P62 (1:1,000; Progen, Heidelberg, Germany), α-LC3 (1:250; Novus, Littleton, CO), and α-multiubiquitin (1:1,000; Enzo, Farmingdale, NY). Secondary antibodies were applied for 1 h at room temperature (Li-Cor α-rabbit, 1:10,000; Li-Cor α-guinea pig: 1:5,000). The PVDF membrane was then imaged on the Li-Cor Odyssey imaging system and quantified using the Li-Cor image studio software. Following the initial probing and analysis, the membrane was blotted with antibodies against β-actin (1:8000; Proteintech, Chicago, IL) or α-tubulin (1:2,500; Sigma, St. Louis, MO) as loading controls.