Supplementary Figure Legends:
Fig. 1 Mutations in dPOMT1 do not affect other markers at NMJ
A. Sample NMJs stained with a-DVGLUT (pre-synaptic compartment), α-Dlg (Postsynaptic
membrane), a-HRP (Presynaptic membrane) and a-FasII (Periactive Zones). There is no change in the
levels or localization of these markers of different compartments of the NMJ of dPOMT1 mutants. B.
Quantification of bouton numbers in WT and dPOMT1 mutants (n=10, p=0.3). C. Gel showing RTPCR performed on WT, w- Crossed to CS and dPOMT1/def mutants.
Fig. 2 Rotated abdomen phenotype of dPOMT1 mutants is rescued by transgenic expression of
POMT1 with actin-Gal4 driver
dPOMT1 mutants (dPOMT1/def=rtp/ df(3L)ED4470) have a rotated abdomen (rt) phenotype (b), which
was rescued using a transgene of dPOMT1 (Rescue = Act-GAL4/UAS-POMT1; rtP/ df(3L)ED4470)
driven by a ubiquitous driver (c), a is WT, CS crossed to w-.
Fig. 3 Pre- or Post-synaptic expression of dPOMT1 is unable to rescue the DGluRIIB levels
A. Sample NMJs of WT, CS crossed to G7-Gal4 or elav-Gal4, dPOMT1 mutant (dPOMT1/def= rtp/
df(3L)ED4470 and Pre- (pre=elavGal4/UAS-dPOMT1;dPOMT1/def) or Post-synaptically
(post=G7/UAS-dPOMT1; dPOMT1/def) driven dPOMT1 transgene in dPOMT1 mutant back ground
stained with a-DGluRIIA (Green), a-DGluRIIA in red and a-HRP (Blue). DGluRIIB levels are
decreased in the dPOMT1 mutant. DGluRIIB levels are not restored by expressing dPOMT1 pre- or
postsynaptically (n=9, p=0.45). B. Quantification of DGluRIIB levels at the NMJs of the genotypes
listed in B.
Supplementary Materials and Methods:
Fly Stocks:
Transgenic flies carrying either pre- or postsynaptic Gal4 Driver (elavGal4 or G7 Gal4) were crossed
into dPOMT1 mutant background. The genotypes were: elavGal4/UAS-dPOMT1; dPOMT1/def (rtp/
df(3L)ED4470) and G7/ UAS-dPOMT1; dPOMT1/def (rtp/ df(3L)ED4470). Other genotypes have been
described in the main materials and methods.
Αntibodies:
α-DVGLUT antibody was used at 1:10,000 (Daniels et al., 2004), mouse α-Discs large (Dlg)
[monoclonal antibody (mAb) 4f3], developed by Corey S. Goodman (Renovis, San Francisco, CA) was
used at 1:2000 and mouse α-Fascilin II (FasII) [monoclonal antibody 3B3], developed by Corey S.
Goodman (Renovis, San Francisco, CA) was used at 1:20 (Lin et al., 1994).
RT-PCR:
RT-PCR was performed using standard protocols and as described in Bloom et al., 2007 (Bloom et al.,
2007) except fly brains were used for RNA extraction and the primer sets were designed to detect
dPOMT1 transcript. RP49 was used as a control.
Supplementary References
Bloom AJ, Miller BR, Sanes JR, DiAntonio A (2007) The requirement for Phr1 in CNS axon tract
formation reveals the corticostriatal boundary as a choice point for cortical axons. Genes
Dev 21:2593-2606.
Daniels RW, Collins CA, Gelfand MV, Dant J, Brooks ES, Krantz DE, DiAntonio A (2004) Increased
expression of the Drosophila vesicular glutamate transporter leads to excess glutamate
release and a compensatory decrease in quantal content. J Neurosci 24:10466-10474.
Lin DM, Fetter RD, Kopczynski C, Grenningloh G, Goodman CS (1994) Genetic analysis of Fasciclin
II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron
13:1055-1069.