Polyclonal rabbit antibody

Inner sections of kidney poles were minced and homogenized in ice-cold RIPA buffer with protease inhibitors (0.15 M NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40, 0.5% deoxycholate). Total protein content was determined using the BCA Protein Assay Kit (Santa Cruz, CA, USA) according to manufacturer instructions. Protein samples (40 micrograms) were separated on a precast NuPAGE 10% Bis-Tris gel (Novex) at 200 v for 45 min. Blots were blocked at 25 °C and incubated with primary antibodies. Then, the membranes were incubated with the secondary antibodies (1:1000 dilutions) and analyzed by normalization against β-actin bands (used as a housekeeping gene). Fibronectin, TGF-β1, and NOX-4 protein levels were detected using polyclonal rabbit antibody from Santa Cruz, CA, used at 1:200 dilutions. Immunoblots are shown in each figure as representative images. Results are shown as the ratio of each band vs. β-actin (fold change of control). Analysis was conducted by using 4–6 animals per group and 3–5 independent experiments for Western blot analysis (Supplementary Figures S1 and S2). Blots were incubated with chemiluminescent substrate ECL reagent (Perkin Elmer, Waltham, MA, USA) by direct exposure on films that were scanned and analyzed by ImageJ software.

Embryos were fixed in 4% PFA (30 min, RT), permeabilised with 0.5% Triton-X100 (30 min, RT) and blocked with 3% BSA. Plk1 phosphorylated at Thr210 was labeled with a polyclonal rabbit antibody (ThermoFisher Scientific; dilution 1:100 in 3% BSA, overnight, 4 °C) and cyclinA2 - with a polyclonal rabbit antibody (Santa Cruz; dilution 1:50 in 3% BSA, overnight, 4 °C), both followed by a secondary anti-rabbit antibody conjugated with Alexa 633 (Molecular Probes, ThermoFisher Scientific; dilution 1:200 in 3% BSA, 1.5 h, RT). Microtubules were stained with mouse anti-tubulin β antibody labeled with FITC (Sigma-Aldrich; dilution 1:50 in 3% BSA, 1.5 h, RT or overnight, 4 °C). DNA was stained with Hoechst 33342 dye (Molecular Probes, ThermoFisher Scientific; 100ng/µl in PBS, 30 min, RT or overnight, 4°C), propidium iodide (Sigma-Aldrich; 3 μg/ml in PBS, 30 min, RT) or chromomycin A3 (Sigma-Aldrich; 10 μg/ml, 30 min, RT or overnight, 4°C). In case of phospho-Plk1 detection, a PhosStop solution (Roche) was added to all stages of immunostaining to inhibit phosphatases.

Nuclear RAD51 foci were visualized and quantified as previously described [26] and used as surrogate markers for induction of DNA DSBs and competent HR DNA repair, respectively. In brief, cells were grown on coverslips (12 mm, Thermo Scientific) and exposed to 10 Gy of IR. After 4–6 hours recovery, the cells were fixed, permeabilized, and immunostained with primary antibodies against RAD51 (polyclonal rabbit antibody, Santa Cruz) and detected with alexa flour 488 (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2- phenylindole (DAPI). The presence of RAD51 foci was evaluated in a minimum of 100 cells in three independent experiments with an automated inverted fluorescence microscope (LEICA–DMI6000 B).