Journal: Nature

Article Title: Astrocyte–neuron subproteomes and obsessive–compulsive disorder mechanisms

doi: 10.1038/s41586-023-05927-7

Figure Lengend Snippet: a . Schematic of the BioID2 biotinylation reaction in the presence of biotin, Mg 2+ , and ATP. Free lysine residues on a protein are biotinylated. b . Diagram of BioID2 biotin labeling in mammalian cells. Proximal (~10 nm) proteins are biotinylated upon the addition of exogenous biotin. c . Schematic of the cell and compartment specific in vivo BioID2 experimental design using adeno-associated viruses (AAVs). d . 40 μm coronal section of mouse brain microinjected in the dorsal striatum with AAV 2/5 GfABC 1 D BioID2-HA (Astro BioID2) and injected subcutaneously with biotin for 7 days. Arrows show the site of Astro BioID2-HA injection and the site of increased biotinylation. e . Western blot analysis of brain unilaterally microinjected with Astro BioID2. Dark bands at 130 kD and 250 kD show the endogenously biotinylated mitochondrial proteins, Pyruvate carboxylase and acetyl-CoA carboxylase. Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 4 mice; two-tailed paired t-test, P = 0.0052). f . Western blot analysis of brain unilaterally microinjected with plasma membrane localized Astro Lck-BioID2. Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 4 mice; two-tailed paired t-test, P = 0.0088). g . Western blot analysis of brain unilaterally microinjected with AAV1 hSyn1 BioID2-HA (Neuro BioID2). Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 4 mice; two-tailed paired t-test, P = 0.015). h . Western blot analysis of brain unilaterally microinjected with plasma membrane localized Neuro Lck-BioID2. Graph depicting the streptavidin signal intensity divided by the β-actin signal intensity for each data point. Black horizontal line depicts the mean, red lines depict the SEM (n = 4 mice; two-tailed paired t-test, P = 0.0023). For blot source data, see Supplementary Fig. 1.

Article Snippet: The following viruses were used: 0.5 μl AAV2/5 GfaABC 1 D-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-Lck-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-Aqp4-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-Cx43-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-Ezr-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-Glt1-BioID2-Linker-BioID2-HA; 0.5 μl AAV2/5 GfaABC 1 D-HA-BioID2-Linker-BioID2-Kir4.1; 0.5 μl AAV2/5 GfaABC 1 D-tdTomato (Addgene, 44332-AAV5); 0.5 μl AAV2/5 GfaABC 1 D-Lck-GFP (Addgene, 105598-AAV5); 0.5 μl AAV2/5 GfaABC 1 D-Aqp4-eGFP; 0.5 μl AAV2/5 GfaABC 1 D-Cx43-eGFP; 0.5 μl AAV2/5 GfaABC 1 D-Ezr-eGFP; 0.5 μl AAV2/5 GfaABC 1 D-Glt1-eGFP; 0.5 μl AAV2/5 GfaABC 1 D-eGFP-Kir4.1; 0.5 μl AAV1 hSYN1-BioID2-Linker-BioID2-HA; 0.5 μl AAV1 hSYN1-Lck-BioID2-Linker-BioID2-HA; 0.5 μl AAV1 hSYN1-eGFP (Addgene, 50465-AAV1); 0.5 μl AAV1 hSYN1-Lck-GFP; 0.5 μl AAV2/5 GfaABC 1 D-Rpl22-HA (Addgene, 111811); and 0.5 μl AAV1 hSYN1-Rpl22-HA.

Techniques: Labeling, In Vivo, Injection, Western Blot, Two Tailed Test, Clinical Proteomics, Membrane