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?
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.
Article of the Year Award: Outstanding research contributions of 2020, as selected by our Chief Editors. Read the winning articles.