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. 2011 Dec 14;6(12):e28710. doi: 10.1371/journal.pone.0028710

Figure 4. Nck and Filamin are necessary for Nephrin-Ship2 interaction.

Figure 4

A . Ship2 is recruited to CD16-Nephrin clusters in the presence of Nck. Human podocytes expressing CD16-Nephrin chimeras and GFP-Ship2 were clustered in the presence of either full length Nck or Nck lacking its Sh2 domain (NckΔSH2). B. Filamin is required for Ship2 recruitment to CD16-Nephrin clusters. Human melanoma cells (M2) which lack Filamin were transfected with CD16-Nephrin chimera and GFP-Ship2 and clustered as described and examined by confocal microscopy. WT-Filamin (wild type) was transfected to rescue the M2 cell line. C. Nephrin activation results in Ship2 phosphorylation. Human podocytes expressing Flag-Ship2 were co-transfected with plasmids encoding the indicated CD-16 chimeric proteins. Cell lysates were resolved by SDS-Page and transferred to a PVDF membrane. The membrane was blotted with a p-Nephrin antibody that recognizes Y5 and Y7 phosphorylation and a p-Ship2 (Y1135) antibody. Scale bar: 10 µm.