Abstract
VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and use visual scoring for immunocytochemistry staining to determine the extent of the antibodies' specificity. Thus, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.
Highlights
Intracellular membrane fusion in the secretory and endocytic pathways relies on SNARE proteins for membrane fusion events
VAMP7 is a clostridial neurotoxin-insensitive v-SNARE that belongs to the “Longin” subfamily: it encompasses an amino-terminal extension, the Longin domain, which acts as an auto-regulatory domain[1]
VAMP7 exocytosis was shown to be regulated by an integrin, FAK, and Src-dependent mechanism in developing neurons[11] and its transport to the cell periphery by VARP, Rab[21] and Kif[57], while retrograde transport depends on LRRK112
Summary
6. Figure 2 and Dataset 2 include a more reliable visual scoring table instead of intensity profile quantification as we agree it might fail to discriminate a typical distribution of VAMP7 (ie: Golgi-like and peripheral vesicles) from an incorrect one (ie: perinuclear-enriched and peripheral diffuse signal, such as ER localization pattern). This total staining does not merely reflect noise and includes non-specific bands and we still think that the index used is quite informative about the Ab quality. Following recent renaming of our institute, affiliation 2 has been amended for authors Beatrice Cholley, Thierry Galli and Sebastien Nola
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