Anti hpx

Spinal cord tissue (1 mm above and below the lesion site, ~ 2 mm length) from the lesion site (T8–T9) was homogenized in RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Tissue lysates were subjected to western blotting using an anti-Arg-1 (Abcam, Cambridge, MA) or anti-Hpx (Abcam, Cambridge, MA) antibody. Protein bands were analyzed and quantified using densitometry and an Image-Pro Plus analysis system (Media Cybernetics, Silver Spring, MD) by normalizing their levels to the levels of GAPDH bands.

PBMCs were lysed for Western blot analysis, as previously described [178 (link)]. Cellular protein amount was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA); cell lysates were denatured in 5X Laemmli sample buffer at 98 °C for 10 min and equal amounts of proteins were separated using 12, 10, or 8% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked in 5% not-fat dry milk in PBS with 0.01% Tween 20 for 1 h at room temperature, and then incubated overnight at 4 °C with the following antibodies: anti-5-LOX (#3289, Cell signaling, Danvers, MA, USA), β-actin (#37100, Cell signaling, Danvers, MA, USA), anti-CP (#98971, Cell signaling, Danvers, MA, USA), anti-Tf (#82411, Abcam, Cambridge, UK), anti-LCN2 (#44058, Cell signaling, Danvers, MA, USA), anti-SOD1 (#2770, Cell signaling, Danvers, MA, USA), and anti-HPX (#124935, Abcam, Cambridge, UK). Subsequently, the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies, and were detected with an enhanced chemiluminescence (ECL) solution (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).