Journal: Vaccines

Article Title: Identification of Potential Amblyomma americanum Antigens After Vaccination with Tick Extracellular Vesicles in White-Tailed Deer

doi: 10.3390/vaccines13040355

Figure Lengend Snippet: Shared proteomic cargo packed within salivary and midgut extracellular vesicles secreted by Amblyomma americanum females fed on white-tailed deer. Extracellular vesicles (EVs) from salivary glands (SG) and midguts (MG) were characterized by mass spectrometry using parallel accumulation–serial fragmentation (PASEF) in a Bruker timsTOF-PRO. ( A ) Venn diagrams representation of unique and shared proteins found within SG-EVs (blue) and MG-EVs (red). ( B ) Enrichment analysis of protein classes shared between A. americanum SG- and MG-EVs. Bars represent the fold enrichment with p -value ≤ 0.05 and false discovery rate (FDR) ≤ 0.05. ( C ) Western blot analysis of heat shock protein 70 (Hsp70) and Calnexin in midgut (MG) and salivary gland (SG) organs (Org) and extracellular vesicles (EVs). Proteins (50 µg for organs and 15 µg for EVs) were separated by SDS-PAGE and transferred into membranes. Numbers next to the ladder represent the size in kilodaltons (kDa). Hsp70 estimated size = ~70 kDa. Calnexin estimated size = ~90 kDa.

Article Snippet: Membranes were probed with rabbit anti-Hsp70 polyclonal antibodies (1:20,000; ProteinTech, Rosemont, IL, USA; #10995-1-AP) or rabbit anti-Calnexin polyclonal antibodies (1:10,000; Millipore sigma; Saint Louis, MO, USA; AB2301) diluted in 10 mL 1X PBS + 25 μL 5% milk + 5 μL Tween 20 and detected with goat anti-rabbit polyclonal antibodies horse-radish peroxidase (HRP) labeled (1:10,000; Abcam; Waltham, MA, USA; AB6721) diluted in 10 mL 1X PBS + 25 μL 5% milk + 5 μL Tween 20.

Techniques: Mass Spectrometry, Western Blot, SDS Page