Anti drp1

LPS was purchased from Sigma (MO, USA). Anti‐Sirt3, anti‐Cyt‐c and anti‐acetylated lysine were obtained from Cell Signalling Technology (MA, USA). Immunofluorescent Anti‐Sirt3 was purchased from Santa Cruz (CA, USA). Anti‐OPA1, anti‐Drp1, anti‐VDAC, anti‐TOM‐20 and anti‐GAPDH were purchased from Abcam (MA, USA). Anti‐Bax and anti‐YME1L1 were purchased from Proteintech (Wuhan, China). Anti‐Flag, Alexa Fluor 488‐ and 594‐conjugated fluorescent secondary antibodies and HRP‐conjugated secondary antibodies were purchased from Antgene (Wuhan, China).

Protein expression was analyzed by Western blot. The total protein (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After the protein is transferred to the polyvinylidene fluoride microporous membrane (Bio-Rad), the membrane is blocked with 5% skimmed milk powder and the primary antibody [anti-Bax, anti-PCNA, anti–β-catenin, anti-Drp1 (1:1,000 dilution, American Abcam), anti-NLRP3 (1:500 dilution, American Abcam), anti-ASC, or anti–caspase-1]; then anti-mouse (anti-rabbit (IgG)) is linked with fluorescein (1: 1,000), and then incubated with an antibody conjugated to anti-fluorescein alkaline phosphatase (1:5,000). The immune complexes were detected with enhanced chemiluminescence reagents. For quantification, the signal optical density was normalized to β-actin by Quantity One image analysis software.

The paraffin-embedded colon sections were deparaffinized, rehydrated, and pretreated with hydrogen peroxide in a PBS buffer. Heat-induced antigen retrieval was performed. The sections were incubated with anti-Drp1 (1:1,000 dilution, Abcam, United States) and anti-NLRP3 (1:100 dilution, Abcam, United States). The sections were incubated with HRP-conjugated secondary antibody, tyramide amplification was performed first, streptavidin-HRP was added, and then the DAB kit was used to show a positive signal. The sections were inspected at 400× magnification and analyzed using NIS-Elements. The following formula was used to calculate the positive content: positive content (PC) = average optical density x positive area.

Cells and heart tissues for western blotting were first lysed in RIPA buffer (Beyotime, Jiangsu, China). Mitochondrial fractions were isolated using the Mitochondria Isolation Kit, based on the manufacturer's instructions (PIERCE, Rockford, IL, USA). Proteins in the lysate (30 μg) were separated using SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked in 5% skimmed milk and 0.1% Tween-20 for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies and dilutions used in this study were anti-SOCS6 (1 : 1000, Abcam, Cambridge, UK), anti-DRP1 (1 : 1000, Abcam), anti-DRP1 (phospho S637) (1 : 500, Abcam), anti-QK (1 : 1000, Cell Signaling Technology, Boston, MA, USA), anti-VDAC1 (1 : 1000, Abcam), and anti-β-actin (1 : 5000; Sigma-Aldrich, St. Louis, MO, USA). Membranes were either probed with horseradish peroxidase-conjugated with goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Final blots were visualized by an Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Amersham, UK). The intensity of protein bands was quantified by ImageJ software.

Whole-cell lysates and mitochondrial fractions were obtained from mice hearts in different groups. Whole cell lysates were prepared as previously described [12 (link)]. Mitochondrial fractions were prepared by using the Mitochondria Isolation Kit (Beyotime Biotechnology, Jiangsu, China). Western blot analysis was performed as previously described [23 (link)]. The following primary antibodies were used: anti-Drp1 (Abcam), anti-VDAC1 (Abcam), and anti-GAPDH (Wuhan Boster Biological Technology).

PBMCs were plated in a 96-well flat-bottom plate and cultured in RPMI-1640 supplemented with 10% FBS at a density of 2 × 10⁶ cells per well. Cells were stimulated with 96-well plates coated with anti-CD3 (Clone: OKT3; 2 µg/mL, Biolegend, San Diego, CA, USA) or PBS control and measured at 4 h and 24 h timepoints at 37 °C/5% CO₂. After stimulation, cells were stained with cell surface markers anti-CD3, -CD4, -CD27, -CD28, -CD45RO and -CD45RA, permeabilized and then stained with anti-GLUT1, anti-OPA1, anti-MFN2 and anti-DRP1 (Abcam, Cambridge, UK). Cell viability was assessed using Live/Dead Aqua (ThermoFisher, Waltham, MA, USA). Cells were analysed using the BD LSRFortessa^TM X-20 cell analyser (BD Biosciences, Franklin Lakes, NJ, USA). Single-stained BD CompBeads were used to define compensation parameters. T-cell subsets were identified using the gating strategy observed in Figure 1.