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SUPPLEMENTAL MATERIAL
Supplemental Figure 1. CaM-KII labeling at individual Club endings. In order to
illustrate the variability in the amount of CaM-KII labeling within the population of CEs, 8
bit images of individual CEs (n=43) were thresholded and total and central areas of each
CE were identified following procedures identical to those used for calculations of the
periphery-center index (see methods). (A) Histogram shows the total amount of CaM-KII
labeling detected at individual CEs, expressed as total CaM-KII pixels above threshold
per total CE pixel area. (B) Histogram shows the amount of CaM-KII labeling detected at
the center of individual CEs, expressed as CaM-KII pixels above threshold at the center
per center CE pixel area. (C) Graph illustrates the relationship between center and total
CaM-KII labeling at individual CEs. Note the strong correlation between these two
measurements.
Supplemental Figure 2. Sequence alignment of the auto-inhibitory region of αCaM-
KII with its fish ortholog and the Cx36 binding sites to αCaM-KII with its orthologs.
(A) The mouse αCaM-KII and teleost (zebrafish) regulatory regions shows high levels of
sequence identity. (B) Sequence alignment between Cx36 cytoplasmic loop (CL) and
carboxyl-terminal (CT) αCaM-KII binding sites (Alev et al., 2008) and its fish homologs
show high degrees of similarity. The Cx36CL binding site, similar to the pseudotarget
binding site of αCaM-KII, shows similarity to amino acids surrounding core residues for
binding. The Cx36CT binding site, highly similar to the pseudosubstrate binding site of
αCaM-KII, reveals a conserved motif and high degree of identity with perch Cx35 and
Cx34.7.
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Supplemental Figure 3. Surgical isolation of hindbrain tissue containing the
Mauthner cells. (A) Mid- saggital view of a goldfish brain showing its different regions:
tectum, cerebellum, vagal Lobe and the hindbrain region in which the M-cells are
located. Gray area represents the portion to be surgically isolated for further material
isolation. (B) Hindbrain region isolated from A. After isolation from the brain, the
cerebellum is removed, leaving a portion of the hindbrain containing the intact M-cells
and CEs, along with other reticulospinal and vestibulospinal neurons. Four to five of
these hindbrain sections were used in each sample for protein quantification.
Supplemental Figure 4. In-silico predictions and alignments of the mammalian Cx36
phosphorylation sites by αCaM-KII with its homologs perch Cx35 and Cx34.7. (A)
CaM-K/CaM-KII/CaM-KIIa (α) phosphorylation sites prediction for Cx36, Cx35 and
Cx34.7 were identified using the Group-based Prediction System Version 2.1 (GPS
v2.1). Four putative αCaM-KII phosphorylation sites were identified previously for Cx36
(Alev et al., 2008) using the group-based phosphorylation scoring method (GPS). Scores
for predicted αCaM-KII phosphorylation sites for Cx36 and their fish counterpart were
higher than the cutoff values (highest threshold) for a high prediction performance. The
prediction of serine 293 as a phosphorylation target site in Cx36 required a lower
threshold (medium), which still offers high accuracy and sensitivity. (B) Sequence
alignment of the regions of Cx36 cytoplasmic loop and carboxyl-terminal containing
phosphorylation sites for αCaM-KII and its fish homologs perch Cx35 and Cx34.7,
demonstrate a high degree of homology. Putative αCaM-KII phosphorylation sites are
outlined in red. Completely conserved amino acids are indicated with “I”, conserved
substitutions with “:”, and semi-conserved substitutions with “.”.