International Journal of Nephrology / 2013 / Article / Fig 6

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 6

NF- B p65 expression, distribution, and EMSA for NF- B consensus in wild type and nephrin-mutated podocytes: (a) WT and Neph podocytes were grown, fractionated, and processed for western blot as described in Section 2. The rabbit anti-p65 (sc-372) polyclonal antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). The sheep anti-histone H1 (H5110) polyclonal antibody was purchased from US Biological (Swampscott, 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). (b) Nuclear extracts from WT and Neph podocytes were prepared as indicated in Section 2. EMSA was performed using NF- B consensus [ ]-labeled double-strand oligonucleotide. The sequence of the probe and the EMSA technique are described in Section 2. The anti-p65 antibody is the same as in part A of the present figure, but 10 times more concentrated (sc-372X) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The figure shows a typical experiment of at least three performed under the same conditions.

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