Tmr conjugated halo ligand

Cells were incubated in DMEM with 100 nM TMR-conjugated Halo ligand (Promega, G8251) for 20 min. The cells were then collected for protein extraction or washed twice with phosphate-buffered saline (PBS) and incubated with amino acid-free and FBS-free DMEM (Wako Pure Chemical Industries, 048–33575) to induce autophagy by starvation or to DMEM with 1 µM oligomycin (Merck Millipore, 495455) and 2 µM antimycin (Sigma-Aldrich, A8674) to induce mitophagy. After the desired incubation time, cells were scraped into ice-cold PBS and centrifuged at 2000×g for 2 min. The cell pellet was then resuspended in lysis buffer (25 mM HEPES-KOH [pH 7.2], 150 mM NaCl, 2 mM MgSO₄, 0.2% n-dodecyl-β-D-maltoside [Nacalai Tesque, 14239–54] with protease inhibitor [Sigma-Aldrich, P8340]). After 20 min of incubation on ice, 1/10 volume of lysis buffer containing benzonase (Merck Millipore, 70664) was added to the cell suspension to a final 1/200 dilution of benzonase. Protein concentration was determined with NanoDrop One spectrophotometer (Thermo Fisher Scientific).

Cells expressing Halo-mGFP were incubated with the full medium containing 100 nM tetramethylrhodamine (TMR)-conjugated Halo ligand (Promega, G8251) for 30 min. After washing twice with PBS, the cells were cultured under DMEM supplemented with FBS (fed condition) or DMEM without amino acids (starved condition) for 6 hours and then collected in ice-cold PBS by scraping on ice. Cell lysates were prepared as described above and subjected to SDS-PAGE. For in-gel fluorescence imaging, the gel was visualized with Odyssey M imaging system (LI-COR). Band intensities were quantified with Fiji.

Halo-LC3 stable expression cells were generated by viral transduction. The pMRX-IP-HaloTag7-LC3 was used for gene transduction. Cells were incubated in DMEM with 100 nM TMR-conjugated Halo ligand (Promega; catalog no.: G8251) for 1 h. Western blotting with an anti-HaloTag antibody (Promega; catalog no.: G9211) was performed to assess the amount of Halo-LC3 and its degradation products. pMRX-IP-HaloTag7-LC3 was a gift from Noboru Mizushima (Addgene plasmid; catalog no.: 184899; http://n2t.net/addgene:184899; Research Resource Identifer: Addgene_184899) (17 ).