Fv10i liv confocal microscopy

Immunofluorescence analysis of LCN2, CHOP and FLAG-tagged LCN2 was performed as described previously^{19 (link),74 (link)}. HeLa cells (2 × 10⁵ cells) on glass (Matsunami Glass Industries, Japan) were incubated with 400 ng ml⁻¹ SubAB or mt SubAB for the indicated time. Cells were fixed with 4% paraformaldehyde (PFA) (Fujifilm WAKO Pure Chemical Corporation) for 30 min. The cells are then rinsed three times with PBS and incubated with blocking buffer (5% goat serum, 0.3% Triton X-100 in PBS) at room temperature for 1 h. To visualize FLAG, PDI, or CHOP, cells were further incubated with primary antibodies in 0.4% BSA/PBS buffer at 4 °C overnight, washed twice with PBS and incubated with anti-mouse 488 (Cell Signaling) or anti-rabbit Cy3 (Rockland) antibodies at room temperature for 1 h in the dark. After washing with PBS three times, cells were mounted on glass slides using Prolong Gold Antifade reagent with DAPI (Thermo Fisher Scientific). The stained cells were visualized by FV10i-LIV confocal microscopy (Olympus). The images were arranged with Adobe Photoshop CS4.

Immunofluorescence analysis was performed as described previously [8 (link), 12 (link)]. Briefly, FLAG-tagged KLHDC7B-transfected HeLa cells (3 × 10⁵ cells/12-well plate) on glass (Matsunami Glass Co. Ltd., Osaka, Japan) were incubated with 400 ng mL⁻¹ of SubAB or mt SubAB. Cells were fixed with 4% paraformaldehyde (PFA), rinsed three times with PBS, and incubated with the blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) at room temperature for 1 h. Cells were further incubated with anti-FLAG antibodies (#014-22383, WAKO) in 0.4% BSA/PBS buffer at 4 °C overnight, washed twice with PBS, and incubated with anti-mouse Cy3 (#ab97035, Abcam, Cambridge, UK) antibodies at room temperature for 1 h in the dark.
After the cells were washed with PBS, they were mounted on glass slides using Prolong Gold Antifade reagent with DAPI (ThermoFisher Scientific). FV10i-LIV confocal microscopy (Olympus, Tokyo, Japan) was used to visualize the stained cells and the images were arranged with Adobe Photoshop CS4.