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Supplementary materials
Supplementary Figure 1
Genotypes in the breeding colony. (A) The diagram shows schematically the five different
genotypes that could occur in the colony. The red bars indicate knockout of Slc9a6 on
Chromosome X. (B) PCR of tissue from mice tails showing the expected band-sizes in base pairs
(bp) for each genotype. For reference see: http://jaxmice.jax.org/pub-cgi/protocols/protocols.
Quantitative Real-Time PCR (Rt-PCR) analysis
Frozen brain tissue (cerebral cortex) was homogenized using MagNA Lyser Green Beads (Roche)
containing TRIzol®Reagent from Invitrogen and the FastPrep® Instrument (Thermo electron
corporation). The supernatant was collected and total RNA was isolated according to the
protocol of TRIzol® Plus RNA Purification Kit (Invitrogen). Any genomic DNA remnants were
removed using TURBO DNase (Ambion; 10 g of total RNA treated with 2 Units TURBO DNase for
30 min. at 37C) and cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems) as per manufacturer's recommendations. The Rt-PCR reactions were
carried out on a StepOnePlus instrument (Applied Biosystems) using 5 ng cDNA, Power SYBR®
Green PCR Master Mix (Applied Biosystems) and 400 nM final concentrations of each primer.
Pre-designed primers were used both for the target gene (Slc9a6 (3 different primer sets)) and
endogenous control (Gapdh). All samples were analyzed in triplicates and with two technical
parallels (3 runs per sample). The relative quantity was calculated using the equation RQ = 2-ΔΔCT,
where RQ is the relative quantity of the target gene. ΔΔCT is the difference in CT-value between
the target gene and the endogenous control minus the difference in CT-values between the
reference gene and the endogenous control. The threshold line was set to 0.1 for all targets in all
runs. Primers used for the Rt-PCR are listed in Supplementary Table 1. The PCR primers were
synthesized by Eurofins MWG Operon (Ebersberg, Germany).
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Supplementary Table 1: Primers used in the Rt-PCR
Target gene (details)
Sequence
Slc9a6 (Primer set 1; exons 1-2)
Forward 5’-ACCGGCCTTGCTATGATTTA-3’
Reverse 5’-TGACTGCACTTCACAGCTCA-3’
Slc9a6 (Primer set 2; exons 6-7)
Forward 5’-GAAAGTGTCCTCAATGACGCGGTT-3’
Reverse 5’-CTGTAACGTCAAAGGTGTGGCTGTTG-3’
Slc9a6 (Primer set 3; exons 12-14,
reverse primer span exons 13-14)
Forward 5’-CAGCACCACACTTCTGATCGTGTT-3’
Reverse 5’-TCCTTTCGTTGTCAGGAACACCCA-3’
Gapdh (endogenous control)
Forward 5’-TCGTCCCGTAGACAAAATGGT-3’
Reverse 5’-CGCCCAATACGGCCAAA-3’
The nucleotide sequences of the PCR primers used to assay gene expression by Real-Time
quantitative PCR are shown.
Antibodies and Reagents
The following primary antibodies were used in immunoperoxidase and immunofluorescence
labeling procedures. GM2 ganglioside monoclonal antibody (mAb) was produced in-house,
hybridoma clone 10-11, kindly provided by Progenics Pharmaceuticals, Inc (Tarrytown, NY). GM3
ganglioside mAb (hybridoma clone DH2) was purchased from GlycoTech (Gaithersburg, MD).
The LAMP2 mAb was developed by J. Thomas August obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and maintained by The University
of Iowa, Department of Biology. Calbindin mAb was purchased from Sigma-Aldrich (St. Louis,
MO). Monoclonal anti-ZebrinII (Brochu et al., 1990) was used directly from spent hybridoma
culture medium (gift from Dr. Richard Hawkes, University of Calgary). X-Gal (5-Bromo-4-chloro3-indolyl--D-galactopyranoside; B9146) was purchased from Sigma-Aldrich. The following
secondary antibodies were purchased from Vector Laboratories for immunoperoxidase labeling:
biotinylated goat anti-mouse IgM, biotinylated goat anti-mouse IgG, and biotinylated goat antirabbit IgG. The following secondary antibodies for immunofluorescence were purchased from
Invitrogen: Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor
546 goat anti-mouse IgM, and Alexa Fluor 555 donkey anti-rabbit IgG. Filipin complex, from
Streptomyces filipinensis, was purchased from Sigma-Aldrich for labeling unesterified cholesterol.
Western blot for tau analysis
Frozen tissue used for western blots was homogenized in ice-cold lysis buffer (50mM Tris-HCl, pH
7.5, 150mM NaCl, 1% Igepal CA-630, 1% sodium deoxycholate, 0.1% SDS) supplemented with
Complete, Mini Protease Inhibitor Cocktail (Roche) and centrifuged as described previously
(Davidson et al., 2009). The supernatants were then collected as soluble protein fractions and
pellets were frozen at -80C as insoluble fractions. Protein concentrations were determined by
BCA protein assay kit (Thermo Scientific). Insoluble pellet fractions were washed with lysis buffer
and further extracted with 2% SDS in sample buffer, sonicated, and centrifuged. For
immunoblotting, samples were analyzed under denaturing conditions by SDS-PAGE and
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transferred to Immuno-blot PVDF membranes (Bio-Rad). Blocking was done with 5% non-fat dry
milk in TBS with 0.1% Tween-20. Primary antibody incubation for PHF-tau (clone AT8; Thermo
Scientific) was performed overnight at 4C and secondary peroxidase labeled antibody was
applied for 1 hour at RT. Protein detection was performed using SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific) and either exposed to film, or developed on a
KODAK 2000R imaging station using KODAK imaging software. An antibody to β-actin (SigmaAldrich) was used as an external loading control. Western blot quantification was performed by
densitometric analysis using ImageJ software. These analyses were represented as PHF-tau/Actin
and data sets were normalized to the WT male (+/y) in each age group.
Supplementary Figure 2
PHF-tau analysis. The figure shows western blot analysis of soluble and insoluble protein
fractions from cerebral tissue of 3 and 8 month old mice probed for PHF-tau (AT8 mAb). We
observed evidence of slight increases in PHF-tau levels in the soluble fraction from Slc9a6
knockout homozygote females (-/-) and hemizygous males (-/y) compared to WT littermate
males (+/y) at both 3 and 8 months of age. Quantification corresponds to the blots shown.
Samples were normalized to the WT males (+/y) at 3 and 8 months time-points. For the insoluble
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protein fraction the major band (~70 kD) was used for quantification. β-actin was used as an
external loading control.
Behavioral analysis methods
The novel object placement test of spatial memory is based on the robust tendency of rodents to
preferentially explore novel objects and was performed essentially as described (Ennaceur and
Meliani, 1992). In Trial 1, mice were given 5 min to freely explore two identical, non-toxic
objects. Mice were then removed to the home cage for a 20 min retention interval, and returned
to the arena for 3 min in which one object was displaced in space. High contrast visual cues were
placed on each wall of the arena and care was taken to ensure the novel position changed both
intrinsic relationship between objects and the relative position to visual cues. The time spent
(sec) exploring the objects was recorded (Trial 2). Data from Trial 2 were expressed as a
preference score—(time exploring novel object/total time exploring) × 100. A preference score
of 50% indicates chance performance. Exploration of the objects was defined as whisking,
sniffing, rearing or touching the object, and approach and obvious orientation to the object
within 3 cm. Novelty-induced exploration of the objects in Trial 1 was also assessed and
expressed as the total time spent exploring both the objects (sec).
Supplementary Table 2: Results from the behavioral assays
Genotype
Result (mean  SE)
P value
Wild type
Slc9a6-/Y
Wild type
Slc9a6-/Y
Wild type
Slc9a6-/Y
35.4  4.5
29.3  5.8
324.7  29.9
324.3  38.6
24.7  2.9
30.8  3.8
0.41
Wild type
Slc9a6-/Y
Wild type
Slc9a6-/Y
68.0  4.4
65.2  6.2
36.0  4.4
30.0  5.7
0.72
Open field*
Center time (sec)
Center track length (cm)
Center entries (#)
1.0
0.22
Object placement (spatial memory)
Preference score (sec)
Total novel object exploration (sec)
0.42
*Two other open field tests, the total track length and number of rears, showed significantly
increased values in Slc9a6-/Y mice (Fig. 8A and B)
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References
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Brochu G, Maler L, Hawkes R. Zebrin II: a polypeptide antigen expressed selectively by Purkinje
cells reveals compartments in rat and fish cerebellum. J Comp Neurol 1990;291:538-52.
Ennaceur A, Meliani K. A new one-trial test for neurobiological studies of memory in rats. III.
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