Mouse anti tfr1 antibody

Protein extraction and Western Blotting were performed as described [51 (link)]. Used antibodies were a rabbit anti-Lcn2 antibody (1:1000; abcam, Cambridge, United Kingdom), a mouse anti-TFR1 antibody (1:1000; Sigma Aldrich), a rabbit anti-ferritin antibody (1:500; Sigma), a rabbit anti-Ho1 antibody (1:1000; abcam), a rabbit anti-Irp2 antibody (1:1000; Novusbio, Littleton, CO, USA) and a rabbit actin antibody (1:500; Sigma Aldrich), and appropriate HRP-conjugated secondary antibodies (1:2000, anti-rabbit; 1:4000, anti-mouse; Dako, Glostrup, Denmark). For quantification, densitometry data were acquired on a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) and analyzed with Image Lab 5.2.1. (Bio-Rad).

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).