Journal: Journal of Virology

Article Title: Rab27a-mediated exosome secretion facilitates classical swine fever virus release and immune evasion

doi: 10.1128/jvi.01488-25

Figure Lengend Snippet: CSFV infection promoted exosome secretion. ( A ) Schematic diagram of exosome isolation. ( B ) NTA was performed to determine the particle size distribution of exosomes isolated from the culture supernatant of CSFV-infected PK-15 cells. ( C ) The morphology of exosomes was visualized using electron microscopy after staining with phosphotungstic acid. ( D ) The total protein concentration of the isolated exosomes was measured using the bicinchoninic acid (BCA) protein assay. ( E ) Exosome proteins were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. ( F ) Western blot analysis was performed to assess the expression of the exosome marker proteins Alix and CD81. ( G ) Schematic diagram of exosome purification. ( H ) CSFV RNA in the purified exosomes was detected by PCR. ( I ) CSFV E2 protein in the purified exosomes was detected by western blot analysis. ( J ) Immunoelectron microscopy confirmed the presence of CSFV E2 protein in the purified exosomes.

Article Snippet: The protein concentration of the isolated exosomes was determined using Coomassie Brilliant Blue staining and a BCA protein assay kit (TargetMol, catalog number C0050).

Techniques: Infection, Isolation, Exosomes, Morphology, Electron Microscopy, Staining, Protein Concentration, Bicinchoninic Acid Protein Assay, SDS Page, Western Blot, Analysis, Expressing, Marker, Purification, Immuno-Electron Microscopy, Polymerase Chain Reaction, Transmission Electron Microscopy

Journal: Journal of Virology

Article Title: Rab27a-mediated exosome secretion facilitates classical swine fever virus release and immune evasion

doi: 10.1128/jvi.01488-25

Figure Lengend Snippet: Rab27a enhanced CSFV replication by promoting exosome secretion. ( A–C ) PK-15 cells were transfected with siNC or siRab27a for 24 hours and then infected with CSFV (MOI = 1) for 48 hours. Exosomes were isolated, and their total protein content was quantified using the BCA assay ( A ) and Coomassie Brilliant Blue staining ( B ). Western blot analysis was performed to assess the expression of exosome marker proteins CD81, Alix, and CSFV E2 protein ( C ). ( D–F ) Cells transfected with PGK-Rab27a or PGK-Flag were infected with CSFV (MOI = 1) for 48 hours. Exosomes were isolated, and their total protein content was quantified using the BCA assay ( D ) and Coomassie Brilliant Blue staining ( E ). Western blot analysis was performed to assess the expression of exosome marker proteins CD81, Alix, and CSFV E2 protein ( F ). ( G ) Schematic representation of the Transwell co-culture experiment. PK-15 cells were infected with CSFV (MOI = 1) and co-cultured with uninfected PK-15 cells in the lower chamber for 72 hours, with or without the addition of an E2 neutralizing antibody. ( H and I ) CSFV RNA levels in lower chamber PK-15 cells were measured by qPCR in the Rab27a knockdown group ( H ) and Rab27a overexpression group ( I ). ( J and K ) IFA was used to detect CSFV infection plaques in lower chamber PK-15 cells in the Rab27a knockdown group ( J ) and Rab27a overexpression group ( K ). qPCR data were normalized to β-actin mRNA levels. Error bar = SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results shown are representative of three experimental repeats.

Article Snippet: The protein concentration of the isolated exosomes was determined using Coomassie Brilliant Blue staining and a BCA protein assay kit (TargetMol, catalog number C0050).

Techniques: Transfection, Infection, Exosomes, Isolation, BIA-KA, Staining, Western Blot, Analysis, Expressing, Marker, Co-culture Assay, Cell Culture, Knockdown, Over Expression, Immunofluorescence