Journal: Scientific Reports
Article Title: Expression of plasma membrane calcium ATPases confers Ca 2+ /H + exchange in rodent synaptic vesicles
doi: 10.1038/s41598-019-40557-y
Figure Lengend Snippet: SV2s do not confer Ca 2+ /H + exchange. ( a ) Expression of SV2 isoforms in synaptic vesicles derived from SV2A/B-DKO and SV2B/C-DKO mice. Equal amounts of vesicle proteins from each genotype (20 µg/lane) were analyzed by western blotting using isoform-specific antibodies. Antibodies used for western blots are indicated at the left of the images. Rabbit polyclonal antibodies against SV2A, SV2B, and SV2C, and a mouse monoclonal antibody that recognizes all SV2 isoforms (pan-SV2) were used. For loading controls, a mouse monoclonal antibody against synaptophysin (Syp) (Cl7.2) was used. Note that expression of the SV2 isoforms was completely abolished in the respective DKO samples. A faint band revealed by the pan-SV2 antibody in the SV2A/2B-DKO sample indicated that SV2C content was much less than that of SV2A or SV2B. The images were cropped from four independent blots for presentation, and the original digital images of the full-length blots are presented in Supplementary Fig. . ( b ) Ca 2+ -induced alkalization in vesicles derived from SV2A/B-DKO (red) compared to wild-type mice (black). 50 µM CaCl 2 was added after vesicles were pre-acidified in the presence of 100 mM KCl. ( c ) Effect of levetiracetam (LEV, 30 µM) on Ca 2+ -induced alkalization in vesicles from wild-type mice. Vesicles were pre-acidified in the presence of 100 mM KCl, and 50 µM CaCl 2 was then added. LEV pre-treatment (red traces) shows little impact on alkalization by Ca 2+ compared to the respective controls without LEV pre-treatment (black traces). ( d ) Effect of levetiracetam (LEV, 30 µM) on Ca 2+ -induced alkalization in vesicles from SV2B/2C-DKO mice. Measurements were performed as in ( c ). LEV pre-treatment (red traces) shows little impact on alkalization by Ca 2+ compared to the respective controls without LEV pre-treatment (black traces). Traces in ( b – d ) are the representative data from two to three measurements in each condition.
Article Snippet: The resulting blots were probed with isoform specific antibodies for SV2s (Synaptic Systems, Germany) or with a pan-SV2 monoclonal antibody (a kind gift from Reinhard Jahn, Göttingen, Germany).
Techniques: Expressing, Derivative Assay, Western Blot
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