Sheep anti rela nfκb

HepG2 at a density of 20,000 cells/cm² treated with 400–1,200 pg/ml of FGF23 were lysed by complete Lysis-M kit (Roche) for cytoplasm and with a second lysis (60 μl of Lysis-M and 10 μl of NaCl 5 M) for the nuclear fractions. The protein lysates were separated on an SDS-PAGE and transferred by electroblotting on a PVDF membrane (Bio-Rad). After blocking, each membrane was incubated with the primary antibody sheep anti-RelA/NFκB 1: 500 (R&D) or anti-TNFα 1:250 (Novus) followed by the HRP-conjugated secondary antibodies 1:200. The products were identified by chemiluminescence (BM Chemiluminescence Western Blotting Kit, Roche). Loading ctrl was conducted with antibodies directed rabbit anti-cofilin 1: 10,000 (SIGMA) and against anti-Histone H3 for Nuclear loading ctrl 1: 1,000 (not shown, Abcam). The images were acquired/analyzed by the Chemidoc XRS instrument and Quantity One software (Bio-Rad).

HepG2 were fixed in cold acetone for 5 min or PFA for 10 min, permeabilized with 0.3% of Triton (Sigma-Aldrich, Milan, Italy) for 30 min and incubated with 1% of bovine serum blocking solution for 1 h. Immunocytochemistry was performed with the primary antibodies sheep anti-RelA/NFκB (R&D systems, Minneapolis, USA), mouse anti-IκBα (Santa Cruz), and rabbit anti-TNFα (Novus, Cambridge, UK) o/n. The secondary fluorescently labeled antibodies were used for 1 h RT: Alexa Fluor 488 donkey anti-sheep IgG, Alexa fluor 488 goat anti-mouse, and Alexa fluor 546 goat anti-rabbit (Invitrogen, Thermofisher, USA). The lack of staining demonstrated the specificity of Ab labeling after substituting the primary antibody with sheep IgG/mouse IgG1/rabbit IgG Isotype ctrl (Invitrogen). For fluorescence in situ hybridization (FISH), the Stellaris RNA protocol for adherent cells using the human AHSG target sequence was applied (Supplementary Table 1). Images were acquired by Zeiss AxioObserver microscope with Apotome system and recorded by AxioVision software 4.8. Human conditionally immortalized podocytes SV1 (HciPodo, University of Bristol, Bristol, UK) were used as negative ctrl. The nuclei were stained with DAPI (Sigma).