Figure 2
In vivo study with FLAG-CLEC-2 protein. (A) Double immunostaining for FLAG and nephrin in kidneys with or without infusion of FLAG-CLEC-2. After infusion of FLAG-CLEC-2, glomeruli were intensely stained for FLAG in nephrin-positive podocytes. Scale bar: 50 μm. (B) Internalization assay using cultured podocytes. Representative images of podocytes stained for FLAG, after 3 h incubation at 37 °C or 4 °C with or without subsequent washing with a stripping buffer. At either temperature, FLAG staining diminished after washing, indicating that FLAG-CLEC-2 was not internalized within the cells. Scale bar: 200 μm. (C) Serpine1 mRNA in the glomeruli (relative amount). Serpine1 mRNA expression was 2.45-fold increased in the mice with FLAG-CLEC-2 infusion. (D) Western blot analysis of glomerular lysate for ERM. Phosphorylation of ERM proteins was 0.65-fold decreased by FLAG-CLEC-2. The images of full-length blots are shown in Supplementary Figs. 7 and 8. Ezrin: 81 kDa, Moesin: 75 kDa, β-tubulin 55 kDa. (E) Dephosphorylation of ERM in podocytes by FLAG-CLEC-2 perfusion. Double immunostaining showed an intense signal for pERM (red) in podocytes labeled by podocalyxin staining (green) in control mice (upper panels). In the mice perfused with FLAG-CLEC-2, some podocytes lack pERM staining. Scale bar: 50 μm. (F) SEM images of the foot processes. Podocytes perfused with FLAG-CLEC-2 exhibited widening of foot processes (arrows) in 18.5% of visual fields. Original magnification: × 8000. Scale bar: 2 μm.
