In-gel fluorescence imaging and immunoblotting

HaloTag processing assay to quantify autophagic activity

1. HeLa cells stably expressing Halo-LC3B (Addgene #184899) are grown in DMEM in 6 cm dishes at 37°C in a 5% CO2 incubator.

2. Remove DMEM.

3. Add 3 mL of DMEM containing 100 nM TMR-conjugated HaloTag ligand (Promega, G8251).

4. The cells are incubated for 20–30 min at 37°C in a 5% CO2 incubator.

5. Remove the ligand-containing DMEM.

6. The cells are washed twice with 4 mL of PBS pre-warmed at 37°C.

7. Add 3 mL of starvation medium (Wako Pure Chemical Industries, 048-33575) pre-warmed at 37°C.

8. The cells are incubated for 4-6 h at 37°C in a 5% CO2 incubator.

9. Remove the starvation medium from the dishes and place the dishes on ice.

10. Add 1 mL of ice-cold PBS.

11. The cells are scraped and centrifuged at 2,000 × g for 2 min at 4°C.

12. Remove supernatants.

14. Freeze cell pellets once at -30°C or -80°C.

15. Solubilize the cell pellets with 120 uL of ice-cold Lysis Buffer (containing 0.2% DDM) for 20 minutes on ice.

16. Divide the cell suspension into 90 uL and the remainder (~30 uL).

17-1. Centrifuge the remainder (~30 uL) of the cell suspension at 10,000 × g for 2 min at 4°C.

17-2. Determine the protein concentration of the supernatant fraction using a NanoDrop One spectrophotometer (Thermo Fisher Scientific).

18. Add 10 uL of Lysis buffer containing Benzonase (Merck Millipore, 70664) to 90 uL of the cell suspension to give a final dilution of 1/200 of Benzonase.

19. Add 100 uL of 2× SDS-PAGE sample buffer to the benzonase-treated cell suspension.

20. The samples are denatured at 55°C for 15 min and then at 98°C for 5 min.

21. Protein concentration is adjusted with 1× SDS-PAGE sample buffer (diluted with the same Lysis buffer).

22. The samples (20 µg protein) are separated by SDS-PAGE.

23. For in-gel fluorescence imaging, the gel is visualized with FUSION SOLO.7S.EDGE (Vilber-Lourmat) immediately after SDS-PAGE.

24. For immunoblotting, proteins are transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, WBKLS0500) using the Trans-Blot Turbo Transfer System (Bio-Rad) and subjected to Western blotting with anti-HaloTag antibody (Promega, N2701).

DMEM: Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, D6546) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, 173012) and 2 mM L-glutamine (Gibco, 25030-081)

DDM: n-dodecyl-β-D-maltoside (Nacalai Tesque, 14239-54)

Lysis buffer: 25 mM HEPES-KOH (pH 7.2), 150 mM NaCl, 2 mM MgSO4, 0.2% DDM, and protease inhibitors (Sigma-Aldrich, P8340)