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For measuring protein activity, we used conditionally immortalized mouse podocytes generated from the ImmortoMouse (Charles River, Wilmington, MA) (Weins et al. 2001).
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Central to this technique is an anti mouse podocyte antibody generated in sheep.
We generated an anti-mouse podocyte antibody that binds to rat and mouse podocytes in vivo.
Mouse podocytes bound and endocytosed both albumin and IgG via receptor-specific mechanisms.
Here, we report that Robo2 is expressed at the basal surface of mouse podocytes and colocalizes with nephrin.
This was accompanied by a retraction of branches of cultured mouse podocytes.
Continuous cultivation of conditionally immortalized mouse podocytes was performed as described by Mundel et al. 1996 [9].
Next, we analyzed whether α-dystroglycan on mouse podocytes could mediate outside-in signaling.
Stabilization of dynamin is thus one approach to rescue podocyte function as shown by gene delivery of cleavage resistant dynamin into mouse podocytes [6].
Sheep anti-podocyte antibody was applied to cultured immortalized mouse podocytes for 30 minutes on ice, and then unbound IgG was washed away.
Fully differentiated immortalized mouse podocytes were incubated with 2 mg/ml podocyte specific IgG on ice for 30 minutes to allow binding.
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