International Journal of Nephrology / 2013 / Article / Fig 3

Research Article

Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by -Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

Figure 3

Phenotypic features of nephrin-mutated podocytes and EMT marker expression in human wild type and nephrin-mutated podocytes: effects of TGF- 1 treatment. (a) The cells were grown as indicated in Section 2. Prior to the TGF- 1 incubation (a) and (e), the cells were starved for 48 hr. The pictures show the cells after 48 hr of TGF- 1 treatment used at 5 ng/mL. The optical images were obtained with a Will (Wetzelar) inverted microscopy at a 10x enlargement. (b) -SMA detection in wild type (WT) and nephrin deprived human podocytes (Neph). The anti- -SMA mouse monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). (c) Detection of fibronectin bound to the membrane fraction and in the cytoplasm. Na-K-ATPase- 1 was used as cell membrane marker to normalize the protein loading. Mouse monoclonal anti-fibronectin (sc-18825), goat anti-Na-K-ATPase- 1 (sc-16041) polyclonal antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (d) Detection of vimentin in the total cell lysate. The goat anti-vimentin (sc-7557) was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (e) Effects of TGF- 1 on vimentin and -SMA expression in the indicated podocyte cells. To quantify the protein bands, we scanned the blots and performed a densitometry analysis, using the software NIH ImageJ v. 6.4 (freeware, NIH, MD, USA). The figure shows a typical experiment of at least three performed under the same conditions.

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