Bradford reagens

2 ml cultivation broth were centrifuged at 20000 g for 10 min at 4℃. The supernatant was discarded and the pellet resuspended in 3 ml 0.1 M NaOH before sonication with a Sonifier®; 250 Cell Disruptor (Branson) (power 70%, duty cycle 40%, power 20 s, pause 40 s, 10 cycles, on ice). The sonicated samples were incubated for 3 h at RT. After centrifugation (20000 g, 10 min, 4℃) the supernatant was used to determine protein concentration with a Bradford assay. Therefore, 20 μl diluted sample (1:10 - 1:100) were added to 1 ml 1:5 diluted Bradford Reagens (Bio-Rad Laboratories) and incubated for exactly 10 min at RT before measuring the absorption on a V-630 UV-Vis spectrophotometer (Jasco, Tokio, Japan) at 595 nm. As standard bovine serum albumin in concentrations from 10 - 100 μg/ml was used.

Protein extracts from BMDMs were prepared using cytoplasmatic lysis buffer: Protein quantity was measured with Bradford Reagens (purchased from Biorad, Hercules, CA, USA), and 15–20 µg was loaded in 10% SDS-polyacrylamide gels. Proteins were separated by electrophoresis at 180 V for about 1 h and blotted on a PVDF-membrane (GE-Healthcare, Chicago, IL, USA) at 100 V. Protein bands were made visible with Posseau-Staining to cut aligned bands. Subsequently, antibody blocking in 5% milk powder prevented unspecific binding of antibodies. The first and secondary antibodies were applied including several washing steps in between TTPS. The protein quantity was evaluated using horse-radish peroxidase mediated chemilumenesce (Bio-Rad, 1:2000, anti-rabbit; 1:4000, anti-mouse; Dako, Glostrup, Denmark). The following antibodies were used: a mouse anti-TFR1 antibody (1:1000; Sigma Aldrich, MW: 100 kDa), a rabbit anti-LCN2 antibody (1:1000; Abcam, Cambridge, UK, MW: 23 kDa), a rabbit anti-FRT antibody (1:500; Sigma Aldrich, MW: 20 kDa), a rabbit anti-iNOS (1:1000; Abcam, Cambridge, UK, MW: 130 kDa), a rabbit anti-FPN1 antibody (1:2000; self-made, MW: 66 kDa), a rabbit anti-PCBP2 (1:1000, antikörper-online.de, 39 kDa) and a rabbit anti-ACTB antibody (1:500; Sigma Aldrich, MW: 45 kDa).