Bca assay

After cultivation with IL‐33 (100 ng/mL) at different time points, total protein extracts were isolated using lysis buffer. Same amounts of protein samples (concentration determined by BCA assay, Serva, Germany) were separated by SDS‐PAGE and electrotransfered onto nitrocellulose membrane Hybond ECL (AppliChem, Germany). Next, membranes were blocked in 4% nonfat milk (Serva) in TBST for 1 hour and subsequently incubated with primary antibodies: mouse anti‐GAPDH, anti‐NF‐κB and anti‐β‐catenin (Santa Cruz Biotechnology) overnight at 4°C. Protein bands were visualized using ECL reagent (Serva, Germany) after membrane incubation with anti‐mouse HRP‐conjugated antibody (Sigma‐Aldrich) during 1 hour at room temperature.

Protein concentration was determined using a BCA assay (SERVA, Germany). The total turnover number is the ratio of the total amount of the yield product in µM to the total amount of enzyme applied in µM.

To test protein binding to cell-free RAD16-I gels, 100 µL of a 0.3% peptide solution was loaded into tissue culture inserts, gelled for 1 h with 10% FBS in PBS or PBS alone (negative control), and then washed with PBS overnight several times. Gels were disrupted in RIPA buffer, and bound protein content was quantified with a BCA assay (39228, Serva). To test collagen binding to RAD16-I gels, 10 μL of a 3 mg/mL collagen solution (A1048301, Gibco) was mixed with 90 μL of 10% sucrose and afterwards mixed with an equal volume of a 0.6% RAD16-I peptide solution. Further, 40 μL of this solution was induced to self-assemble with PBS for 1 h, and then washed with PBS overnight several times. Gels were disrupted by vortexing in 2x SDS sample loading buffer (LC2676, Thermo Fisher, Waltham, MA, USA), mixed with β-mercaptoethanol at 10% final concentration and boiled for 5 min at 95 °C. Samples (20 μL) were loaded in 10% Tris-Glycine pre-cast protein gels (XP00105BOX, Thermo Fisher) and run by applying 120 V for 90 min. Finally, gels were stained with Coomassie blue and distained overnight.