Ddit3

All reagents were purchased from Sigma‐Aldrich (St. Louis, MO, USA), Wako (Osaka, Japan), or Takara (Kyoto, Japan), unless otherwise stated. Anti‐DYKDDDDK antibody (Wako), anti‐TMEM160 antibody (Invitrogen, Carlsbad, CA, USA), anti‐VDAC1 antibody (Calbiochem‐Novabiochem, San Diego, CA, USA), anti‐HSPA9 (Sigma‐Aldrich), anti‐TOMM22 (Tom22) antibody [4 (link)], anti‐HSPD1 antibody (Sigma‐Aldrich), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (anti‐GAPDH) antibody (Novus Biologicals, Littleton, CO, USA), anti‐LONP1 antibody (Sigma‐Aldrich), and Total OXPHOS Rodent WB Antibody Cocktail (Abcam, Cambridge, UK) containing anti‐ATP5A, UQCRC2, and MTCO1 antibodies were used to immunostain the cells and/or in immunoblot analysis. The anti‐TOMM40 (Tom40) and anti‐TOMM20 antibodies were obtained previously [5 (link), 6 (link)]. Anti‐ATF4, ATF5, and DDIT3 antibodies were purchased from Proteintech (Rosemont, IL, USA).

Cells were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). Proteins were separated using Bolt Bis-Tris Plus gels (Thermo Scientific) and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h and then incubated with antibodies against ASNS (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ATF4 (1:1000, Santa Cruz), DDIT3 (1:1000, Proteintech, Rosemont, IL, USA), VEGFA (1:500; proteintech), and β-actin (1:10,000, Santa Cruz). Membranes were incubated with horseradish peroxidase-conjugated horse anti-rabbit or anti-mouse IgG (GenDEPOT, Katy, TX, USA). Protein bands of protein were visualized using an ECL kit (DoGenBio, Seoul, Republic of Korea) and detected using an X-ray film.