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. 2023 Jul 13;12:RP86972. doi: 10.7554/eLife.86972

Figure 4. Biochemical and genetically assisted electron microscopy (EM) validation of the vesicular localization of SLC6A17.

(A) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-HA-2A-iCre mice. More details in Figure 1—figure supplement 1D. (B) Higher magnification views of co-immunostaining by anti-Syp and anti-HA antibodies in the hippocampus. Scale bar = 10 μm. (C) Immunoisolation of SLC6A17-HA containing vesicles by the anti-HA antibody coated on magnetic beads. SLC6A17-HA fraction was positive for Syp, Syt1, Syb2, v-ATPase, VGluT1, VGluT2, and vGAT, but negative for GluT4 and transferrin receptor. (D) Further purification of the LP2 fraction by sucrose gradient showed that SLC6A17-HA was co-immunoisolated with Syp, Syt1, and VGluT1, but not PSD95, ERp72, EEA1, SNAP23, PSMC6, or STX6. (E) A schematic diagram illustrating the APEX2-based labeling strategy with AAV-PHP.eb virus mediated SLC6A17-APEX2 overexpression in vivo. SLC6A17 was fused in-frame to three repeats of the HA tag, a V5 tag, and APEX2. (F) Representative EM image of synaptic vesicles (SVs) labeled by SLC6A17-APEX2 and darkness distribution of DAB-positive and DAB-negative SVs in sections of Slc6a17-APEX2 mouse brains. Red arrow pointing to APEX2 labeled SVs. White arrow pointing to unlabeled SVs.

Figure 4—source data 1. Original files of the full raw unedited blots for Figure 4C and D.
Figure 4—source data 2. Uncropped blots with the relevant bands labeled for Figure 4C and D.

Figure 4.

Figure 4—figure supplement 1. Immunoisolation and immunohistochemistry of Slc6a17-HA mice.

Figure 4—figure supplement 1.

(A) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-HA-2A-iCre KI mice. (B) Confocal images of the hippocampal region in Slc6a17HA/+ mice after immunocytochemistry with anti-Syp and anti-HA antibodies. Scale bar = 50 μm. (C) With fractions of whole brains by differential centrifugation, SLC6A17-HA was co-immunoisolated with synaptic markers including Syp, Syt1, Syb2, and V-ATPase, but not with PSD95. (D) Analysis of synaptic vesicles (SVs) isolated by anti-HA beads from Slc6a17-HA-2A-iCre mice and from WT mice. 23 markers for organelles were analyzed to confirm SV specificity, and absence of markers of lysosome, Golgi, mitochondria, ER, endosome, proteasome, and postsynaptic cytoplasmic membrane. Scale bar = 10 μm.
Figure 4—figure supplement 1—source data 1. Original files of the full raw unedited blots for Figure 4—figure supplement 1C and D.
Figure 4—figure supplement 1—source data 2. Uncropped blots with the relevant bands labeled for Figure 4—figure supplement 1C and D.
Figure 4—figure supplement 2. Electron microscopy (EM) confirmation of the synaptic vesicle (SV) localization of SLC6A17-APEX2.

Figure 4—figure supplement 2.

(A, C, E, G) Different views of SLC6A17-APEX2 overexpression. (B, D, F, H) Normal distribution of SV electron density in the corresponding images on the left.