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Supplementary Figure S1 Genotyping of SV2B knockout (KO) mice. (a) Depletion of
SV2B gene was verified by genomic PCR. Wild-type (wt) band is 789 bp, and mutant
type (mt) band is 520 bp. (b) Expression of SV2B mRNA was not detected by RT-PCR
analysis of the kidney cortex of KO mice. (c) The band for SV2B was not detected by
Western blot analysis of brain lysates of KO mice. (d) Staining with the anti-SV2B
antibody was not detected in glomeruli of KO mice.
Supplementary Figure S2 Findings of the absorption test for the anti-CD2AP antibody.
Normal rat kidney sections were stained with the anti-CD2AP antibody pre-absorbed
with the fusion protein of the GST only (left) or the anti-CD2AP antibody pre-absorbed
with the GST fusion protein of full length CD2AP (center) or normal rabbit serum
(NRS) (right). Although the specific staining was detected in the section stained with
the anti-CD2AP antibody pre-absorbed with the fusion protein of the GST only (left),
no specific signals were detected in the sections stained with the anti-CD2AP antibody
pre-absorbed with the GST fusion protein of full length CD2AP (center) or NRS (right).
Supplementary Figure S3 Western blot findings of the kidney cortex lysates of WT and
SV2B KO mice. A positive band of approximately 85 kDa was detected with the antipPI3K (p85) and PI3K (p85) antibody in the kidney cortex lysates of WT and KO mice.
No differences in the expression levels of phosphorylated PI3K p85 were detected
between KO and WT mice. No band was detected with normal rabbit serum (NRS).
Supplementary Figure S4 Representative Immunofluorescence images of primary
cultured cells of glomeruli from WT and SV2B KO mice. Staining of CD2AP in primary
cultured cells of glomeruli from WT mice was detected at the processes of cells (arrow),
while that of CD2AP in primary cultured cells of glomeruli from KO mice was not
detected at the processes. Although staining of neurexin in WT cells was detected at
the processes of cells (arrow), that of neurexin in KO cells was clearly lower when
compared with WT cells.
Supplementary Figure S5 Schematic diagram of SV2B functions in the podocyte. SV2B
regulates the expression of synaptotagmin on neuron-like vesicles in the podocyte. The
SV2B and synaptotagmin complex interacts with neurexin at the slit diaphragm. The
SV2B/synaptotagmin/neurexin complex is involved in the proper localization of CD2AP,
nephrin, and NEPH1. SV2B is also involved in the exocytosis for laminin.
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Fukusumi et al.
Supplementary Table S1
Characteristics of WT and SV2B KO mice.
Supplementary Table S2
Sequences of primers used in real-time RT-PCR analysis.
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