Anti alpk1

The renal tissue samples from all animals were fixed with 4% paraformaldehyde for 3 days and embedded in paraffin. The 5 μm sections were stained with haematoxylin and eosin (H&E), specific antibodies and Masson's trichrome for histological evaluation and scoring. The protein signal was detected using the appropriate primary antibody amplifier, horseradish peroxidase (HRP)‐conjugated polymer and DAB chromogen/substrate. The slides were immunostained with anti‐NFkB p65 (50‐fold of dilution; Abcam), anti‐ALPK1 (100‐fold of dilution; GeneTex Inc), anti‐CCL2 (50‐fold of dilution; Abcam) and anti‐CCL5 (50‐fold of dilution; Abcam). The tubular injury was scored based on the degree of tubular necrosis, loss of brush border, cast formation and tubular dilatation, as in previous reports.19, 20 The scoring standard was as follows: 0‐normal kidney; 1‐minimal injury (<5% involvement); 2‐mild injury (5%‐25% involvement); 3‐moderate injury (25%‐75% involvement); and 4‐severe injury (>75% involvement).

For detection of ALPK1, the cells were lysed with RIPA buffer containing protease inhibitors on ice for 10 minutes. The extracts were centrifuged at 14 500  g for 5 minutes at 4°C. Protein samples were separated by SDS‐PAGE gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies as indicated and peroxidase‐conjugated secondary antibodies and protein signals detected by enhanced chemiluminescence reagent. The primary antibodies used were as follows: anti‐ALPK1 (1000‐fold of dilution; GeneTex Inc), anti‐lectin (1000‐fold of dilution; Bioorbyt), anti‐NFkB (1000‐fold of dilution; Abcam) and anti‐Actin (3000‐fold of dilution; GeneTex Inc).