Anti tfr1 antibody

Cells were first rinsed in ice-cold phosphate-buffered saline (PBS), pH7.2, and lysed in 1% NP-40, 125 mM Tris, pH6.8, containing a protease inhibitor cocktail (Roche, Basel, Switzerland). For western blotting, 35–50 µg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, SOD1, SOD2, SDHB and ISCU (Abcam, Cambridge, MA, USA), anti-catalase (Santa Cruz Biotech., Santa Cruz, CA, USA), anti-XOD antibody (ProteinTech Inc., Chicago, IN, USA), anti-TfR1 antibody (Zymed, San Francisco, CA, USA), anti-FXN, IRP1, and IRP2 (polyclonal, raised from rabbit). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, UK). Quantification of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/).

Proteins from lysates were prepared and resolved by 12% SDS-PAGE at 100 V, transferred for 1.5 h at 250 mA onto Nitrocellulose membranes, and analysed by immunoblotting as described previously^{56 (link)}. The information for primary antibodies is as follows: anti-ferritin (cat# 69090), SdhA (cat# 137040), and SdhB (cat# 178423) from Abcam (Cambridge, MA, USA), anti-Xod (cat# 55156-1-AP), citrate synthase (cat# 16131-1-AP), aconitase 2 (Aco2) (cat# 11134-1-AP), Ndufs1 (cat# 12444-1-AP), Ndufs3 (cat# 15066-1-AP), Uqcrc1 (cat# 21705-1-AP), and Uqcrfs1 (cat# 1843-1-AP) from Proteintech Group Inc. (Chicago, IN, USA), anti-Actin (cat# BM0627) from Boster (Wuhan, China), anti-Tubulin (cat# T0198) from Sigma-Aldrich (St. Louis, MO, USA), anti-TfR1 antibody (cat# 136800) from Zymed (San Francisco, CA, USA), anti-IscU, Fech, Irp1, and Irp2, anti-Fxn (self-made)^{31 (link)}, anti-CytC (cat# 1896-1) from Epitmics (Burlingame, CA, USA), anti-Vdac (cat# 4661S) from Cell Signaling Technology Inc (Shanghai, China).