anti timm23  (Proteintech)

Journal: Brain, Behavior, & Immunity - Health

Article Title: Immunoglobulin G complexes from post-infectious ME/CFS, including post-COVID ME/CFS disrupt cellular energetics and alter inflammatory marker secretion ☆

doi: 10.1016/j.bbih.2026.101187

Figure Lengend Snippet: IgG from ME/CFS patients induces mitochondrial fragmentation . A. Representative confocal microscopy images show mitochondrial architecture in primary HUVECs expressing stable GFP within mitochondria and exposed to 1 μg/mL purified IgG from ME/CFS, PCS-CFS, and MS patient and control sera. One representative image is shown for each condition. B. Quantification of average mitochondrial surface area in primary HUVECs exposed to 1 μg/mL purified IgG from ME/CFS (n = 39), PCS-CFS (n = 15), MS (n = 20) patients, and healthy controls (n = 41). At least 10 cells were imaged per sample, derived from 3 replicates. In the left panel, each point represents the mean surface area across all images for each subject. In the right panel, each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Left) HD vs ME/CFS, ∗∗∗P = 0.0004. HD vs PCS-CFS, ∗P = 0.0164. HD vs MS, ns P = 0.8676. (Right) HD vs ME/CFS, ∗∗∗∗P < 0.0001. HD vs PCS-CFS, ∗∗P = 0.0067. HD vs MS, ns P = 0.4794. C. Gender-based separation of average mitochondrial surface area from the above experiment. Each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Female) HD vs ME/CFS, ∗∗P = 0.0018. HD vs PCS-CFS, ∗P = 0.0173. HD vs MS, ns P = 0.4938. (Male) HD vs ME/CFS, ns P = 0.0976. HD vs PCS-CFS, ns P = 0.5058. HD vs MS, ns P = 0.4462. D. Capillary-based automated Immunoblot analysis of Mitofilin, Drp1, TIMM23, PLD6, Miga1, LC3β, Pink1 protein levels in HUVEC cells 36 h after exposure to 1 μg/mL of purified IgG from ME/CFS (n = 12), PCS-CFS (n = 12), and MS (n = 11) patient and control (n = 12) sera. Vinculin was used as a loading control. E. Fold change values from the above immunoblot analysis were derived from densitometric analysis of bands, normalized to the same value for vinculin. Two-tailed Mann-Whitney U test. (Drp1) HD vs ME/CFS, ns P = 0.0684. HD vs PCS-CFS, ∗P = 0.0387. HD vs MS, ns P = 0.7859. (PLD6) HD vs ME/CFS, ∗∗∗∗P < 0.0001. HD vs PCS-CFS, ∗∗∗P = 0.0007. HD vs MS, ns P = 0.6075. (Timm23) HD vs ME/CFS, ns P = 0.1402. HD vs PCS-CFS, ∗P = 0.0317. HD vs MS, ∗P = 0.0106. (Miga1) HD vs ME/CFS, ∗P = 0.0284. HD vs PCS-CFS, ∗P = 0.0449. HD vs MS, ns P = 0.1896. (LC3β) HD vs ME/CFS, ns P = 0.0684. HD vs PCS-CFS, ns P = 0.1600. HD vs MS, ns P = 0.6075. (Mitofilin) HD vs ME/CFS, ∗∗P = 0.0056. HD vs PCS-CFS, ∗∗P = 0.0029. HD vs MS, ns P = 0.3164. F. Immunoblot analysis shows a decreased amount of intracellular IgG heavy and light chains in HUVECs pretreated with Fc blocker before the exposure to 1 μg/mL purified IgG from ME/CFS patients or control sera for 12 h. Vinculin was used as a loading control. G. Schematics of IgG cleavage assay. H. Immunoblot analysis shows the presence of intracellular IgG heavy and light chains in HUVECs after exposure to 1 μg/mL purified IgG, purified Fab fragments, and Fc fragments from ME/CFS patients or control sera for 12 h. Vinculin was used as a loading control. Fc-specific secondary antibody was used (second panel) to detect the full-length heavy chain of IgG and cleaved Fc fragments. I. Quantification of average mitochondrial surface area in primary HUVECs exposed to 1 μg/mL purified IgG or Fab Fragment or Fc fragment from 5 ME/CFS, and 5 controls. In the upper panel, each point represents the mean surface area across all images for each individual. In the lower panel, each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Upper Panel) HD vs ME/CFS IgG, ∗P = 0.0317. HD vs ME/CFS Fab, ns P = 0.8413. HD vs ME/CFS Fc, ns P = 0.6905. (Lower Panel) HD vs ME/CFS IgG, ∗P = 0.0130. HD vs ME/CFS Fab, ns P = 0.5572. HD vs ME/CFS Fc, ns P = 0.3662. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Assays were performed using antibodies: Mitofilin antibody (#ab245764, Abcam), Drp1 antibody (#sc-101270, Santa Cruz Biotechnology), TIMM23 (#sc-514463, Santa Cruz Biotechnology), PLD6 antibody (#ab237612, Abcam), FAM73A (Miga1) Antibody (# PA553611 , Invitrogen), LC3β antibody (#18725-1-AP, Proteintech), PINK1 antibody (#NB100-644, Novus Biologicals).

Techniques: Confocal Microscopy, Expressing, Purification, Control, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Western Blot, Cleavage Assay