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. 2012 Aug;23(8):1351–1363. doi: 10.1681/ASN.2012030271

Figure 2.

Figure 2.

Characterization of AA-4E-BP1 transgenic rats. (A) Expression of human 4E-BP1 by immunofluorescence (red) in podocytes as identified by WT1-positive nuclear immunofluorescence (green) in homozygous transgenic rats (upper panels) and wild-type rats (lower panels). The right two panels show DAPI-stained nuclei for the same images. n=3. Ab, antibody. Original magnification, ×160 (B) Western blots of protein extracts from isolated glomeruli of wild-type (WT), heterozygous (Het), and homozygous (Homo) rats. Note that the heterozygous and homozygous glomeruli contain hypophosphorylated lower-molecular-weight bands not seen in wild-type glomeruli (upper blot), and antihuman 4E-BP1 antibodies recognize bands in the heterozygous and homozygous rat glomerular extracts not present in wild-type extracts (middle blot). β-actin is shown as a reference loading marker. n=2 per experiment, with two experiments performed. (C) SEM (top panel) and TEM (bottom panel) images of homozygous AA-4E-BP1 rats, illustrating normal podocyte structure at 100-g body weight. n=4 for SEM and n=2 for TEM. Original magnification ×2500. (D) Western blots of wild-type, heterozygous, and homozygous isolated glomerular extracts demonstrating increased S6 protein and phospho-S6 in heterozygous and homozygous rats compared with wild-type rats; there is no obvious difference in expression of podocyte products (nephrin, podocin, GLEPP1, and vascular endothelial growth factor [VEGF]). β-actin is shown as a reference loading marker. n=2 per experiment, with two experiments performed. (E) Glomerulus from a 100-g homozygous AA-4EBP1 rat developed for phospho-S6 immunofluorescence demonstrating predominant expression of phospho-S6 in podocytes as confirmed by WT1-positive (green) nuclei. The right panel shows DAPI-stained nuclei in the same image. n=3. Original magnification ×160. (F) Hematoxylin and eosin (H and E)–stained sections of rat renal cortex from an 11-month-old heterozygous AA-4EBP1 rat (left panel) versus the cortex from an 11-month-old wild-type Fischer 344 rat (right panel), demonstrating that ESRD had developed in the transgenic rat. n=7. Original magnification ×50.