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Volume 85, Issue 1, Pages (January 2014)

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1 Volume 85, Issue 1, Pages 124-133 (January 2014)
Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis  Moumita Barua, Eric Shieh, Johannes Schlondorff, Giulio Genovese, Bernard S. Kaplan, Martin R. Pollak  Kidney International  Volume 85, Issue 1, Pages (January 2014) DOI: /ki Copyright © 2014 International Society of Nephrology Terms and Conditions

2 Figure 1 Pedigree of family FG-HI, sequencing, and multisequence alignment. (a) Pedigree for family FG-HI. Affected individuals are indicated in gray. Individuals who are heterozygous for the variant PODXL p.L442R are denoted by ‘+,’ whereas those without the mutation are denoted by ‘-’. Individuals without a notation were not tested because no sample was available. (b) Next-generation sequencing reads across podocalyxin (PODXL) aligned to the reference genome in Integrative Genome Viewer (IGV). The antisense strand is indicated as reference. (c) Sanger sequencing confirming the PODXL variant in all affected individuals where DNA was available. The sense strand is indicated. (d) Multisequence alignment showing conservation of the affected amino acid residue, p.442L indicated with an ‘*’. Kidney International  , DOI: ( /ki ) Copyright © 2014 International Society of Nephrology Terms and Conditions

3 Figure 2 Focal and segmental glomerulosclerosis (FSGS)-associated genetic variants do not alter the stability of podocalyxin (PODXL) protein. Madin–Darby canine kidney (MDCK) cells were cotransfected with green-fluorescent protein (GFP) and equal amounts of either wild-type PODXL plasmid, PODXL plasmid containing an FSGS-associated variant (p.L442R to p.K515R), or PODXL plasmid containing a control variant from the Exome Sequencing Project (p.G112S to p.M490V). After 24h, the cells were lysed, and the lysates were immunoblotted for PODXL (upper panel) or GFP (lower panel). *Amino acid is deleted. Kidney International  , DOI: ( /ki ) Copyright © 2014 International Society of Nephrology Terms and Conditions

4 Figure 3 The focal and segmental glomerulosclerosis (FSGS)-associated p.L442R variant induces the formation of podocalyxin (PODXL) dimers. (a) Madin–Darby canine kidney (MDCK) cells transfected with either wild-type (Wt) or p.L442R PODXL plasmid were mock treated or treated with either PNGase F, neuraminidase, or a combination of O-glycosidase and neuraminidase. No differences were observed before or after deglycosylation patterns in both PODXL wild-type and p.L442R mutant on western blotting. (b) MDCK cells were transfected with either PODXL wild-type plasmid or PODXL p.L442R plasmid. The cells were lysed after 24h in either mild lysis buffer (Tris-buffered saline+1% NP-40) or lysis buffer with ionic detergent (RIPA). PODXL was detected using an anti-PODXL antibody. Arrows indicates upper form. Kidney International  , DOI: ( /ki ) Copyright © 2014 International Society of Nephrology Terms and Conditions

5 Figure 4 The focal and segmental glomerulosclerosis (FSGS)-associated p.L442R variant does not alter the subcellular localization of podocalyxin (PODXL). Madin–Darby canine kidney (MDCK) cells transfected with PODXL wild-type (Wt) or p.L442R plasmid. PODXL was labeled with an anti-PODXL antibody (red), F-actin was labeled with phallodin (green), and nuclei were labeled with Hoechst (blue). (a) Horizontal slices of confocal stacks. (b) Vertical slices of confocal stacks. (c) Transfected MDCK cells were surface biotinylated, and cell surface proteins were pulled down using streptavidin beads. Total PODXL and biotinylated PODXL were visualized by western blot. Kidney International  , DOI: ( /ki ) Copyright © 2014 International Society of Nephrology Terms and Conditions

6 Figure 5 The focal and segmental glomerulosclerosis (FSGS)-associated L442R variant does not alter colocalization of podocalyxin (PODXL) with ezrin at the apical surface. Madin–Darby canine kidney (MDCK) cells cotransfected with ezrin and either wild-type (Wt) or p.L442R PODXL plasmid were fixed. PODXL was labeled with an anti-PODXL antibody (red) and GFP-tagged ezrin was labeled with anti-GFP antibody (green). Bar = 20μm. Kidney International  , DOI: ( /ki ) Copyright © 2014 International Society of Nephrology Terms and Conditions

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