Membrane-bound LC3 detection by immunoblotting

UV-induced apoptotic thymocytes were added to BMDMs cultured in 6-well plate at a ratio of 5:1. After incubating for 1.5 h, BMDMs were washed 3 times with DPBS and harvested with CellStripper. Cells from each well were lysed with 70 μL RIPA lysis buffer (Millipore) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche) for 30 min on ice. Lysates were then centrifuged at 12,000g for 10 min and supernatant were transferred to a fresh tube. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fisher). Equal amount of protein were mixed with 4X Bolt LDS sample buffer and 10X reducing reagent dithiothreitol (Novex Bolt Sample Reducing Agent, 10X). Samples were heated for 10 min at 60 °C and centrifuge at 12,000g for 30 s before loading to a 16% Tris-glycine gel. Proteins were then electro-transferred to a 0.45 μm (or 0.2 μm) PVDF membrane (Thermo Scientific). After blocking with 5% milk, the membrane was incubated with rabbit anti-LC3B primary antibody (ab48394, Abcam) overnight at 4 °C. The membrane was then washed for 3 times in TBST and incubated with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) for 1 h at room temperature. After the final wash to remove unbound antibodies, the protein expression was detected by SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged using ChemiDoc Imaging System (Bio-rad). Band intensity was quantified using the software ImageJ.