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?
-Catenin, TCF-4, LEF-1, p53, and pRB distribution and expression in wild type and nephrin-mutated podocytes. The cells were lysated, fractionated and processed for western blot as described in Section 2. (a) Distribution and expression of -catenin in cell membranes and nuclear compartment. The goat anti--catenin (sc-1496) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (b) Expression of LEF-1 and TCF-4 in nuclear compartment. The goat anti-LEF-1 (sc-8591) and anti-TCF-4 (sc-8631) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (c) Phosphorylated and total p53 distribution and expression in nucleus and cytoplasm. The mouse monoclonal anti-p53 IC12 (no. 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no. 9284) antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). (d) Phosphorylated-RB expression in total cell lysate. The rabbit anti-phospho-RB (ser780) (no. 9307) antibody was purchased from Cell Signaling Technologies (Boston, MA, USA). 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). Where the relative amount of protein is indicated as generic “ratio” instead of ratio to a specific housekeeping protein, it is because the protein quantization referred to the different cell compartment housekeepers indicated at the bottom of each blot. The figure shows a typical experiment of at least three performed under the same conditions.
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