Anti tomm20 antibody

U2OS cells were transfected with FLAG-LotB or FLAG-LotC by GeneJuice transfection reagent (Merck) for 24 hr and fixed by 4% paraformaldehyde for 20 min. After fixation, cells were permeabilized and blocked by 0.1% saponin and 1% BSA in PBS for 1 hr at room temperature. Cells were incubated with anti-Flag antibody (Sigma and Cell Signaling), with either anti-calnexin antibody (Abcam), anti-TOMM20 antibody (Abcam), or anti-GM130 (BD Transduction Laboratories) at 4°C overnight. Alexa Fluor 488 and Alexa Fluor 546 (Invitrogen) secondary antibody were incubated for 1 hr at room temperature. Images were acquired by the Zeiss LSM780 microscope system with 63 × 1.4 NA oil immersion objective and further analyzed by Zeiss Zen microscope software.

ST cells and HEK293A cells were purchased from China Institute for Veterinary Drug and cultured in Dulbecco’s Minimal Essential Medium (DMEM; Hyclone) with 10% fetal bovine serum (FBS; Sigma). All cells were maintained in 37°C 5% CO₂ incubator. The PSV was isolated from PSV-infected piglets in Hunan Province, China, and kept in our laboratory. Z-VAD-FMK, ZIETD-FMK, and Z-LEHD-FMK obtained from Beyotime. The PSV VP1 protein-specific monoclonal antibody was prepared by our laboratory. The GAPDH, Bax, Bcl-2, and Cyt c antibodies were purchased from ABclonal. Antibodies against caspase-3 (D3R6Y), caspase-8 (1C12) and caspase-9 (Human Specific) were obtained from Cell Signaling Technologies. The antibody against PARP1 was purchased from Beyotime. The anti-Tomm 20 antibody were purchased from Abcam. The antibody against Flag was purchased from Sigma. HRP-goat anti-mouse lgG, HRP-goat anti-rabbit lgG, Alexa Fluor 549-goat anti-mouse lgG, and Alexa Fluor 488-goat anti-mouse lgG antibodies were obtained from Proteintech.

HeLa cells were cultured on a Lab-Tek Chamber glass slide. Fluo-4-AM (5 μM), MitoSOX (5 μM), and DCF (5 μM) (Thermo Fisher, MA, USA) were added in HBSS buffer (0.49 mM MgCl₂, 0.41 mM MgSO₄, 5.33 mM KCl, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 137.93 mM NaCl, 0.34 mM Na₂HPO₄, 5.56 mM D-glucose, with or without 1.26 mM CaCl₂) (Gibco-Thermo Fisher, MA, USA) and incubated for 10 min. Afterward, the cells were washed once with HBSS, and peptides were treated with HBSS. Time-lapse images were obtained using an Argon laser scanning confocal microscope (Leica TCS SP5 Microsystems, Wetzlar, Germany) at 10-s intervals for 10 min at an excitation of 488 nm for Fluo-4 and MitoSOX, or at 496 nm for DCF. As mitochondrial markers, MitoTracker Green (0.5 μM) and MitoTracker Red (0.1 μM) were preincubated (green for 30 min and red for 2 min), anti-TOMM20 antibody (Abcam, Cambridge, UK) was used for immunocytochemistry and immunofluorescence (ICC/IF), and mito-DsRed2 were transfected with Effectene (Qiagen, Hilden, Germany) on the day before confocal microscopy. Other details are available in the manufacturer’s instructions.