Tmr conjugated halotag ligand

For immunoblotting, the following antibodies were used: mouse monoclonal anti-HSP90 (61049, BD Transduction Lab, San Jose, CA) and anti-WIPI2 (MABC91, Sigma-Aldrich) antibodies and rabbit polyclonal anti-p62 (PM045, MBL, Tokyo, Japan), anti-ATG2A (PD041, MBL), anti-ATG2B (15131-1-AP, Proteintech, Rosemont, IL), anti-ATG16L1 (M150-3, MBL) and anti-WIPI4 (19194-1-AP, Proteintech) antibodies. Rabbit polyclonal anti-LC3 antibody was described previously (69 (link)). Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (111-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA) and anti-rabbit immunoglobulin G (111-035-144, Jackson ImmunoResearch Laboratories) antibodies were used as secondary antibodies. As described above, 100 nm bafilomycin A₁ (B1793, Sigma-Aldrich) was applied. Tetramethylrhodamine (TMR)-conjugated HaloTag ligand (G8251, Promega) and SF650-conjugated HaloTag ligand (A308-02, Goryo Chemical, Tokyo, Japan) were applied at concentrations of 100 and 200 nm, respectively. LipidTOX Red (H34476, Thermo Fisher Scientific, Waltham, MA) was used for the observation of lipid droplets.

The cultured cells were washed twice with 1 mL of development buffer without Ca²⁺ or Mg²⁺ (DB−; 5 mM Na₂HPO₄ and 5 mM KH₂PO₄), plated at a density of 5 × 10⁶ cells mL⁻¹ on a 35-mm dish (IWAKI) filled with 1 mL of DB (5 mM Na₂HPO₄, 5 mM KH₂PO₄, 2 mM MgSO₄, 0.2 mM CaCl₂), and incubated for 5–6 at 21 °C. During the last 30 min, 3 nM TMR-conjugated HaloTag ligand (Promega) was added to the cell suspension for observation under TIRFM and 2 μm ligand for confocal microscopy.