Supplementary Figure 2: Expression levels of the deglycosylation mutants of SV2A, 2B, and 2C in neurons.
From: N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A
Hippocampal/cortical neurons cultured from SV2A(-/-)SV2B(-/-) mice were infected with lentiviruses that express either WT SV2A, 2B, and 2C, or indicated deglycosylation mutants. Cell lysates were harvested and subjected to immunoblot analysis. Actin served as a loading control. The lentiviral vector contains two separated synapsin promoters, with one driving expression of SV2 and the other driving expression of GFP. Thus, GFP served as an internal control for viral infection. Immunoblot signals of SV2 were quantified, normalized using GFP signals, and compared between WT and deglycosylation mutants. The same amounts of viruses were used for WT SV2A and SV2A-N573A (panel a), and for WT SV2C and SV2C-N559A (panel c). The deglycosylation mutation has no effect on SV2A and modestly reduced the expression level of SV2C in neurons. However, it severely reduced the expression level of SV2B. As shown in panel b, even with 10-fold more viruses, SV2B-N516A expression was still drastically lower than WT SV2B. The data are presented as mean ± S.D., n = 3.
