Journal: Frontiers in Immunology
Article Title: Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins
doi: 10.3389/fimmu.2025.1408992
Figure Lengend Snippet: Characterization of NcGA7-binding proteins. (A) Silver staining for SDS–PAGE to identify NcGRA7-binding proteins by an anti-FLAG immunoprecipitation assay using 293T cells transfected with empty plasmid (Mock, Lanes 1 and 2) or NcGRA7 cDNA fused with a FLAG tag (Lanes 3 and 4). Enriched or exclusively detected proteins are shown as five bands, while the other three bands (#1, #2 and #3) were excluded from the MS analyses. M: molecular marker. Each experiment (biological replicate) was repeated two times. (B, C) Western blots of the fractions eluted from the anti-FLAG immunoprecipitate were analyzed by immunoblotting using antibodies against FLAG (B) , XPOT, XPO1, SLC25A13, DNAJA1, UQCRC2, PHB1, and PHB2 (C) . 293T cells transfected with empty plasmid (E) or NcGRA7 cDNA fused with a FLAG tag (G7) at 20 h posttransfection and HFFs infected with the parental strain Nc1 of N. caninum (WT) and NcGRA7-complemented (7c) parasites at 40 h postinfection were used. Each experiment (similar biological replicate) was repeated two time (B, C) .
Article Snippet: Anti-FLAG M2 mouse monoclonal antibody (Sigma–Aldrich), anti-XPOT (Exportin-T) rabbit antibody (A303-972A, Bethyl, Montgomery, TX, USA), anti-XPO1(CRM1) rabbit antibody (A300-469A, Bethyl), anti-SLC25A13 rabbit antibody (GTX109001, GeneTex, Irvine, CA, USA), anti-DNAJA1 rabbit antibody (A304-516A, Bethyl), anti-UQCRC2 rabbit antibody (GTX114873, GeneTex), anti-PHB1 rabbit antibody (GTX101105, GeneTex), anti-CoxIV rabbit monoclonal antibody (3E11, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH rabbit monoclonal antibody (14C10, Cell Signaling Technology), anti-Mfn1 rabbit antibody (13798-1-AP, Proteintech, Rosemont, IL, USA), anti-AIF mouse monoclonal antibody (sc-13116, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mtHSP70 mouse monoclonal antibody (JG1, Invitrogen, Waltham, MA, USA), anti-VDAC1 rabbit antibody (4866, Cell Signaling Technology), anti-PHB2 mouse monoclonal antibody (sc-133094, Santa Cruz Biotechnology), anti-Histone H3 rabbit antibody (9715S, Cell Signaling Technology), anti-phospho-NF-κB p65 rabbit monoclonal antibody (93H1, Cell Signaling Technology) and anti-NFκB p65 rabbit polyclonal antibody (sc-109, Santa Cruz Biotechnology) were used for western blot and indirect fluorescent antibody test (IFAT) in this study.
Techniques: Binding Assay, Silver Staining, SDS Page, Immunoprecipitation, Transfection, Plasmid Preparation, FLAG-tag, Marker, Western Blot, Infection