Ab63929

The kidney sections were ground using a homogenizer or the cells were scratched and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with proteinase inhibitor (Roche, Switzerland) for 30 min on ice. Protein concentration was quantified using the Bicinchoninic acid assay (Beyotime, Shanghai, China). Protein samples (50 μg) were used to perform immunoblotting as described previously^{42 (link)}. The specific primary antibodies used were: anti-GSDME (ab215191, Abcam, 1:1000), anti-caspase-3 (9662, CST, 1:1000), anti-cleaved caspase-3 (9661, CST, 1:1000), phospho-NF-κB p65 (3033, CST, 1:1000), NGAL (ab63929, Abcam, 1:1000), KIM-1 (AF1817, R&D systems, 1:1000), and anti-GAPDH (5174, CST, 1:10000). Peroxidase-conjugated goat anti-rabbit secondary antibodies (A0277, Beyotime, Shanghai, China) were used for immunoblotting.

Immunocytochemical staining of MSC for expression of antimicrobial peptides was performed as described previously (9 (link), 16 (link)). Briefly, 1 X 10⁴ cells were seeded on round coverslips (Chemglass Life Sciences, Vineland, NJ) placed within 24-well-cell culture plates overnight, then fixed with 4% paraformaldehyde (Fisher Scientific, Hampton, New Hampshire) for 10 min, washed with PBS and permeabilized with 0.1% Triton X-100(Sigma-Aldrich,). Slides were then blocked using 5% v/v normal donkey serum and then incubated with primary antibodies, diluted appropriately. Antibodies used for these studies included surfactant protein D antibody (ab203309), lipocalin-2 antibody (ab63929), beta 2 defensin antibody (ab9871), hepcidin antibody (ab134790), and cathelicidin antibody (ab180760), all obtained from Abcam (Cambridge, MA). Specificity controls for immunostaining included purified IgG antibodies from non-immune rabbits or goats. Following primary antibody incubation, slides were washed and incubated with secondary antibodies, either donkey anti-rabbit or donkey anti-goat, both conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania) and the slides were then counter-stained with DAPI to visualize cell nuclei. Image capture for fluorescence staining was done using an Olympus IX83 spinning disk confocal microscope.

Cells were seeded into six-well plates at a density of 2 × 10⁵ cells per well and grown overnight. Cells in wells were transfected with 1000 ng of plasmids including pDCUg-NT, pDCUg-FL, pDM-NT and pDM-FL, respectively. At 48 h post transfection, the whole-cell extracts were prepared using a Total protein Extraction kit (BC3711, Solarbio, China) according to the manufacturer’s instructions. The protein lysates (20 μg/sample) were resolved by SDS-PAGE and the target proteins were detected with Western blot (WB) using the antibodies as follows: GAPDH Rabbit monoclonal antibody (ab181602, Abcam, UK) (1:10,000 dilution), SLC40A1 Rabbit polyclonal antibody (ab58695, Abcam, UK) (1:10,000 dilution), and Lipocalin-2 Rabbit polyclonal antibody (ab63929, Abcam, UK) (1:10,000 dilution). The second antibodies were IRDye® 800CW Goat anti-Rabbit IgG (C80118-05, Licor) (1:10,000 dilution). The blots were detected and fluorescence intensity was quantified with the Odyssey Infrared Fluorescence Imaging System (Licor) and Odyssey software.

Protein extraction and Western blotting were performed as described previously (Hoffmann et al., 2021 (link)). The following antibodies were used: a rabbit FPN antibody [1:2000; Eurogentec, custom made (Petzer et al., 2020 (link))], a mouse TFR1 antibody (1:1000; Sigma Cat# SAB4300398), a rabbit FT antibody (1:500; Sigma), a rabbit NGAL antibody (1:1000 Abcam, ab63929), a rabbit NRF2 antibody (1:1000, Abcam, ab31163), and a rabbit actin antibody (1:500; Sigma Cat# A2066). Appropriate HRP-conjugated secondary antibodies (1:2000, anti-rabbit; Dako Cat# P0399 1:4000, anti-mouse; Dako Cat# P0447) were used. For quantification, densitometry data were acquired on a ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with Image Lab 5.2.1. (Bio-Rad Laboratories GmbH).

Western blot was performed using a lysate of kidney cortex or cultured PTECs. The primary antibodies used were as follows: anti-CPT1α (ab128568, Abcam), anti-KIM1 (ab47635, Abcam), anti-NGAL (ab63929, Abcam), anti-E-cadherin (610181, BD Company), anti-AQP1 (ab168387, Abcam), anti-vimentin (ab92547, Abcam), anti-fibronectin (F3648, Sigma Aldrich), anti-collagen I (1310-01, Southern Biotech) and anti-Tubulin (T6074, Sigma Aldrich). Western blot was performed three times independently. Quantification was completed by scanning and analyzing the intensity of hybridization signals by using NIH Image program.

Total protein was extracted from injured spinal cord tissue stored at − 80 ℃ by using radioimmunoprecipitation assay (RIPA) lysis and extraction buffer that included a protease inhibitor cocktail. The concentration of protein was determined by the BCA method. Equal amounts of protein (30–50 µg) from each sample were separated using 4–20% SurePAGE™ Gels (Genscript) and transferred to nitrocellulose (NC) membranes (EMD Millipore Corp). The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated overnight at 4 °C with the following specific primary antibodies: rabbit anti-iNOS (18,985–1-AP, Proteintech, 1:1000), rabbit anti-C3 (ab200999, Abcam, 1:1000), rabbit anti-GFAP (SAB4300647, Sigma, 1:1000), rabbit anti-JAK2 (3230, Cell Signaling Technology, 1:1000), rabbit anti-pJAK2 (AF3022, Affinity, 1:1000), rabbit anti-STAT3 (12,640, Cell Signaling Technology, 1:1000), rabbit anti-pSTAT3 (ab76315, Abcam, 1:1000), rabbit anti-Lcn2 (ab63929, Abcam, 1:1000), and anti-β-actin (66,009–1-Ig, Proteintech, 1:1000). After incubation with the corresponding secondary antibodies (1:2000) for 1 h at room temperature, the membranes were scanned with ECL-Plus Reagent (Millipore) and observed under an Amersham Imager 600 (General Electric). The band intensity was analyzed by using ImageJ software (NIH).

Immunofluorescence was performed to calculate the number of positive cells and to evaluate the activation states of microglia and astrocytes. Sections were rinsed three times in phosphate-buffered saline (PBS) for 5 min each at room temperature and then blocked with 1% donkey serum containing 0.3% Triton X-100 for 30 min. The sections were then incubated with primary antibodies overnight at 4 °C and with appropriate secondary antibodies at 37 °C for 2 h the next day. Finally, to label the nuclei, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: anti-NeuN (1:100, Abcam, ab177487), anti-glial fibrillary acidic protein (GFAP) (1:400, ab4674, Abcam), anti-ionized calcium binding adapter molecule 1 (Iba1) (1:500, Abcam, ab178846), anti-iNOS (1:500, Abcam, ab49999), anti-C3 (1:300, Abcam, ab200999), and anti-Lcn2 (1:200, Abcam, ab63929). Images were obtained under a fluorescence microscope (BX51, Olympus).