Extended Data Fig. 13: Characterization and validation of Crym-BioID2 for proteomics.
From: Crym-positive striatal astrocytes gate perseverative behaviour
a, Schematic of the astrocyte-specific Crym-BioID2 AAV construct and its corresponding GFP control. Proximal proteins within ~10 nm are biotinylated after the addition of exogenous biotin. b, Representative IHC images of striatal astrocytes from mice microinjected with GfaABC1D-Crym-BioID2 AAVs and treated with biotin. The tissue was immunostained with anti-HA antibody (red), S100β (green), or with a fluorophore conjugated streptavidin probe (grey). Right panel shows endogenous µ-crystallin (magenta) co-localized with S100β (green). c, Representative images of immunostained mouse striatum injected with astrocyte-specific Crym-BioID2 and then treated with biotin for 7 days. Panel shows the immunostaining pattern with S100β as an astrocyte cell marker. d, As in c but with NeuN as a neuronal cell marker. e, Bar graphs depicting the percent of S100β positive or NeuN positive cells with HA expression in a 40x magnification field of view. Black portion of the bar graphs show percent co-localization. (n = 8 fields of view at 40x magnification from 4 mice). f, Western blot analysis of brain unilaterally microinjected with Crym-BioID2. Graph depicts the streptavidin signal intensity divided by the β-actin signal intensity for each data point. (n = 4 mice; two-tailed paired t-test). For gel source data, see Supplementary Fig. 1. g, Table shows the number of peptides and proteins found in the astrocyte-specific Crym-BioID2 proteomics experiments. Each row shows the number of proteins after filtering. h, Bar graph shows the relative µ-crystallin protein expression in LFQ from each neuron and astrocyte subcompartment BioID2 proteomics experiment from ref. 48 and the present study with Crym-BioID2. i, Images of PLA puncta for µ-crystallin and Map2 in tdTomato (tdT) positive astrocytes in a control experiment where the negative PLA probe was omitted. j, Same as i but for µ-crystallin and Usp9x. Average data are shown as mean ± s.e.m. and all statistics are reported in Supplementary Table 5.
