Goat anti mouse lcn2 antibody

After isolation, the livers of WT or lcn2^−/− mice were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound (OCT; Tissue-Tek O.C.T. Compound, Wayne, PA, USA), and then frozen. The frozen livers were sliced into 6 μm-thick sections using a cryostat microtome (Leica Biosystems, Wetzlar, Germany), and the sections were mounted on aminopropyltriethoxysilane-coated slides. The slides were washed with PBS, pH 7.4, to remove the OCT, and incubated with 3% bovine serum albumin (v/v in PBS) for 1 h at room temperature for blocking. Then, the slides were incubated with goat antimouse Lcn2 antibody (R&D systems, Minneapolis, MN, USA) and rat antimouse F4/80 antibody (BioRad, Hercules, CA, USA) at 1:100 in PBS overnight at 4 °C. Alexa 594-conjugated donkey antigoat antibody (ThermoFisher Scientific, Waltham, MA, USA) and Alexa 488-conjugated goat antirat antibody (ThermoFisher Scientific, Waltham, MA, USA) were used as secondary antibodies diluted at 1:100 in PBS. The nuclei were stained with ProLong Gold antifade reagent with 4′,6-diamino-2-phenylindole (DAPI; ThermoFisher Scientific, Waltham, MA, USA). The fluorescent signals were imaged at a 200× magnification using a Zeiss confocal microscope (LSM 510, Zeiss Laboratories, Oberkochen, Germany). Representative images are shown.

Western blottings were performed as previously described26 (link) using the following: a goat anti-mouse LCN2 antibody (R&D systems) at 1:1,000, a rabbit anti-mouse CHOP antibody (Santa Cruz Biotechnology) at 1:500, a rabbit anti-mouse p-c-JUN antibody (Cell Signaling Technology) at 1:1,000, a rabbit anti-mouse pPERK antibody (Cell Signaling Technology), a rabbit anti-mouse ATF4 antibody (Santa Cruz Biotechnology) at 1:500, a rabbit anti-mouse p-eIF2α antibody (Cell Signaling Technology) at 1:1,000 and a rabbit anti-mouse cleaved caspase 3 antibody (Cell Signaling Technology) at 1:1,000, followed by either a rabbit HRP-conjugated anti-goat antibody at 1:10,000 (Dako) or a donkey HRP-conjugated anti-rabbit antibody at 1:10,000 (Amersham). Mouse monoclonal anti-α-tubulin or anti-β-actin antibody (Sigma) was used as loading control. The uncropped versions of western blottings are shown in Supplementary Fig. 15.

Dynabeads Protein G (10 µL, Cat# 10007D, Thermo) was incubated with 0.2 μg of LCN2 mAb (Cat# MAB18571, R&D) in 100 µL PBS for 60 min at room temperature [57 (link)]. For the competitive interaction between LCN2 mAb and mouse LCN2 protein, LCN2 mAb bound on the Dynabeads was incubated with 0.06 μg of recombinant mouse LCN2 protein (Cat# 1857-LC, R&D) and mouse LCN2 mAb epitope peptide (YNVTSILVRDQDQGCRYWIRT, synthesized by AnaSpec, Fremont, CA, USA) in 10X, 100X, or 1000X molar ratio of LCN2 protein in 100 µL PBST for 16 h at 4 °C. For the competitive interaction between LCN2 mAb and human LCN2 protein, recombinant human LCN2 protein (Cat# 1757-LC, R&D) and human LCN2 mAb epitope peptide (YNVTSVLFRKKKCDYWIRT, synthesized by AnaSpec) were used. The immunoprecipitated mouse or human LCN2 protein was detected by Western blot using goat anti-mouse LCN2 antibody (Cat# AF1857, R&D; 1:1000 dilution in Western Antibody Dilution Buffer) or goat anti-human LCN2 antibody (Cat# AF1757, R&D; 1:1000 dilution in Western Antibody Dilution Buffer), and an EasyBlot anti-goat IgG kit (Cat# GTX228910-01, GeneTex).