Anti nik

The total proteins were extracted from kidney tissues as described elsewhere (Ostmann et al., 2013 (link)). Protein concentrations were measured using a BCA protein assay kit (Keygen). Equal amounts (50 μg) of lysate proteins were separated on 8% gel (for detection of IKKα and p-IKKα, respectively), transferred onto PVDF membrane, blocked with 5% nonfat dry milk in TBST buffer (TBS, pH 7.5, 0.1% Tween 20) for 1 h and then incubated overnight at 4°C with rabbit polyclonal anti-IKKα antibody (1:300, diluted in TBST, Santa Cruz Biotechnology), rabbit polyclonal anti-p-IKKα antibody (1:200, diluted in TBST, Santa Cruz Biotechnology), anti-p52 (1:500, Santa Cruz Biotechnology), anti-p-p52 (1:150, Santa Cruz Biotechnology), anti-RelB (1:500, Cell Signaling); anti-p-RelB (1:300, Cell Signaling), anti-NIK (1:500) from Santa Cruz Biotechnology or rabbit polyclonal anti-β-actin antibody (1:500, diluted in TBST, BIOS). On the following day, after extensive washing with TBST buffer, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000, diluted in TBST, KeyGEN Biotechnology) for 2 h, then developed with the use of an enhanced chemiluminescence system (ECL kit, KeyGEN Biotechnology) and captured on light-sensitive Kodak imaging film.

Proteins were extracted from the ultracentrifugation pellets and separated on a denatured SDS-polyacrylamide gel before transfer to a polyvinylidene difluoride membrane (0.45 um; Millipore, Bedford, MA, USA). Afterwards, the membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour. The membranes were probed with primary anti-TRAF3 (1:200, Santa Cruz, sc-949), anti-PTEN (1:1000, Cell Signaling, #9559), anti-p65 (1:200, Santa Cruz, sc-372), anti-NIK (1:200, Santa Cruz, sc-7211) or anti-NFATc1 (1:1000, Cell Signaling, #8032), respectively. To check the amount of proteins transferred to nitrocellulose membrane, GAPDH was used as control and detected by an anti-GAPDH (1:500; Santa Cruz, sc-25778) antibody. The membranes were then washed three times with TBST and incubated for 1 hour with HRP-conjugated secondary antibodies (1:2000; Santa Cruz, sc-2004). The antigen-antibody complexes were visualized using SuperSignal^® West Dura Extended Duration Substrate (Thermo Scientific, Prod # 34075). Supplementary Fig. 7 depicts uncropped western blots.

Western blots were performed as described previously41 (link). Primary antibodies were rabbit polyclonal anti-Fn14 antibody (1:1000, Cell Signaling, Hertfordshire, UK), rabbit polyclonal anti-p100/52 (1:500, Cell Signaling), rabbit polyclonal anti-CCL21 (1:500, Santa Cruz), goat polyclonal anti-CCL19 (1:500, R&D Systems), RANTES (1:500, Acris) and anti-NIK (1:500, Santa Cruz). Since chemokines were assessed in cell lysates, part of the protein would have been expected to be secreted, especially at later time points. Blots were then probed with anti-α-tubulin (1:2000, Sigma) and levels of expression were corrected for minor differences in loading.