Trip br1

Antibodies against the following proteins and tags were from commercial sources: TRIP-Br1, Enzo Life Science (RRID:AB_2052741); XIAP, R and D Systems (RRID:AB_2215008); AC1 (RRID:AB_2223098), AC5/6 (RRID:AB_2257941), ubiquitin (RRID:AB_778730), GAPDH (RRID:AB_10847862), and β-actin (RRID:AB_626632), Santa Cruz Biotechnology; AC2, Novus Biologicals; V5 (RRID:AB_2556564) and transferrin receptor (RRID: AB_86623), Life Technologies; HA (RRID:AB_2314672), Covance; ubiquitin (linkage-specific K27, K48, and K63), Abcam; Phospho-CREB, Cell Signaling Technology; NeuN, Millipore; and Na-K-ATPase (α subunit) (RRID:AB_258029) and FLAG M2 (antibody RRID:AB_439685 and affinity gel RRID:AB_10704031), Sigma-Aldrich.

Cells were centrifuged, washed in ice-cold PBS buffer, and then lysed in RIPA lysis buffer. The amount of protein was quantified using a protein assay kit (Bio-Rad, Korea). Each sample was subjected to SDS-PAGE and transferred to an Immobilon Transfer Membrane (Millipore, Cat#IPVH00010). The filter was incubated with each corresponding antibody, and immunodetection was carried out using the PowerOpti-ECL Western blotting detection reagent (Bio-Rad). The antibodies used in this study were purchased as follows: Bax (Santa Cruz, sc-20067), cyclophilin A (CypA)(Enzo Life Sciences, BML-SA296), HIF-1α (Cell Signaling, Cat#3716S), HMBG1 (Enzo Life Sciences, ALX-210-964), LC3 (Enzo Life Sciences, ALX-803-082), p62/SQSTM1(Cell Signaling, Cat#5114), RIP3 (Santa Cruz, sc-47368), TRIP-Br1 (Enzo Life Sciences, ALX-804-645), XIAP (Cell Signaling, Cat# 2042) and γ-tubulin (Santa Cruz, sc-7396).

Cells (5 × 10⁴ cells) were grown on a sterilized confocal dish (Coverglass-Bottom Dish, SPL. Cat#100350) for 24 h. Cells were incubated with Mitotracker (Invitrogen, #M22426) for 30 min and fixed with 4% formaldehyde for 30 min. These cells were then washed with PBS twice and incubated with TRIP-Br1 (Enzo Life Sciences, ALX-804-645) or LC3 (Cell Signaling Technology, #2775S) antibodies overnight at 4°C. These primary antibodies were detected with an anti-mouse IgG H&L (Alexa Fluor® 568) (Abcam, #ab175473) or an anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, #ab150077). Nuclei were stained with DAPI (Invitrogen, Cat#P36931) for 10 min after washing with PBS. Colocalization between fluorophores was analyzed using ImageJ software (ver. 1.51u; National Institutes of Health, USA). For lysosomal confocal imaging, cells were incubated with 100 nM Lysotracker (Invitrogen, Cat# L12492) in a phenol-free medium for 90 min. Confocal images were obtained using a Zeiss confocal microscope (Nikon A1 confocal). Image manipulation and merging were performed using appropriate tools of the ImageJ software.