Figure S1. Validation of antibodies used
(A) Anti-Lynx1 antibody (sc-23060) 1:1000 detected on cortical samples from wild-type (WT) and
Lynx1 knock-out (KO) mice as well as rat. There is a clear band around 12 kDa, corresponding to
the expected molecular weight of Lynx1, which is not present in Lynx1 KO mice. (B) Anti-β2
antibody (#834, provided by Cecilia Gotti) 1:1000 detected on cortical samples from rat and human
samples as well as α7 and β2 WT and KO mice. There is a clear band around 50 kDa,
corresponding to the expected molecular weight of β2, which is not present in β2 KO mice. (C)
Anti-Ly6H antibody ((H00004062-M01) 1:1000 detected on 0.1 μg GST-tagged recombinantly
expressed human Ly6H protein (Abnova #H00004062-P01) and 40 μg samples of rat cortex, rat
cerebellum and human cortex. A clear band around 20 kDa is detected in rat and human cortex, but
not in rat cerebellum. This is in line with the previous demonstration that Ly6H is expressed in
human cortex, but not cerebellum (Horie et al., 1998). A clear band around 45 kDa is detected in the
recombinant protein sample, which is in line with an extra 25 kDa being added due to the GST tag.
Figure S2. Validation of synaptosome preparation
Representative western blot images of frontal cortical and hippocampal tissue fractionated using
differential centrifugation into a pellet 1 (P1, nuclear) and pellet 2 (P2, crude synaptosome) fraction.
Enrichment of synaptosomes in P2 is confirmed by emrichment of the synaptic protein Syntaxin
(Anti-Syntaxin, MAB336) in the P2 fraction compared to P1 and the total homogenate (Hom)
Figure S3. Validation of primary neuronal cultures
Protein levels in cortical tissue from an adult male rat (CTX), in rat cortical primary neurons
cultured for 7 days in vitro (Neuron DIV7), in rat primary microglia cultured for 15 days in vitro,
and in rat primary astroglia cultured for 18 days in vitro
Glial fibrillary acidic protein (GFAP) is an astrocyte-specific protein. There is clear staining of
GFAP in adult cortex and astroglial cultures, but an almost complete lack of GFAP staining in
neuronal and microglial cultures.
MAP2 is a neuron-specific protein. There are two MAP2 bands in adult cortex, corresponding to the
high molecular weight isoforms MAP2a and c and the low molecular weight isoform MAP2c.
There is clear MAP2c staining in neuronal cultures, and no MAP2 staining in astro- or microglial
cultures. MAP2c has previously been shown to be expressed mainly in immature neurons (Chung,
Kindler, Seidenbecher, & Garner, 1996), which is in line with our data.
Antibodies used for detection were Anti-GFAP (#G9269) 1:5000 and Anti-MAP2 (#MM03) 1:500.
Figure S4. Validation of primary astroglia cultures
Rat primary astroglia stained with: (A) the microglia-specific protein OX-42 (mouse, BD#550299,
1:50) combined with AlexaFluor488 Donkey Anti-mouse 1:300 (green) and the astrocyte-specific
protein glial fibrillary acidic protein GFAP (rabbit, Abcam#ab7260, 1:500) combined
withAlexaFlour533 Goat Anti-rabbit 1:500 (red). The micrograph was taken at 10x magnification
from a confluent culture, scale bar= 10 μm. Or (B) the neuron-specific protein MAP-2 (mouse,
Sigma #4403, 1:500) combined with AlexaFluor488 Donkey Anti-mouse 1:500 (green) and GFAP
(rabbit, Abcam#ab7260, 1:500) combined with AlexaFlour533 Goat Anti-rabbit 1:500 (red). The
micrograph was taken at 20x magnification from a non-confluent culture, scale bar= 10 μm.
Counting of immunopositive cells show that the astroglia cultures contain ≥99% GFAP-positive
cells.
A
B
References
Chung, W. J., Kindler, S., Seidenbecher, C., & Garner, C. C. (1996). MAP2a, an alternatively
spliced variant of microtubule-associated protein 2. Journal of neurochemistry, 66(3), 1273–
81.
Horie, M., Okutomi, K., Taniguchi, Y., Ohbuchi, Y., Suzuki, M., & Takahashi, E. (1998). Isolation
and characterization of a new member of the human Ly6 gene family (LY6H). Genomics,
53(3), 365–8.