Bca kit

Dulbecco’s modified eagle medium (Art. No.10–013-CVRC, Corning, New York, NY, USA); β-sitosterol (Batch No. Y22A10C85758, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), fetal bovine serum (Art. No.04–007-1a, Biological Industries, Kibbutz Beit Haemek, Israel), phosphate buffer saline (PBS; Art.No. WH0112201 911 XP, Procell, Wuhan, China), pancreatin (Art. No.143188, Biosharp, Hefei, China), DMSO (Tianjin Fuyu Fine Chemical Co., Ltd., Wuching District, China), MTT (Art. No. M8180, Beijing Suleibao Technology Co., Ltd., Beijing, China), a BCA kit (Lot No.20210922, Bio-Swamp Life Science Lab, Wuhan, China), an ECL high-sensitivity chemiluminescent solution kit (Batch No.: GC 10AA0033, Biological Engineering Co., Ltd., Shanghai, China), β-actin (Batch No.: F200040, Abways Technology, Shanghai, China), VEGFA (Batch No. 83 m8093, Affinity Biosciences, Beijing, China), PTGS2 (Batch No. 86F4760, Affinity Biosciences), VEGFR2 (Batch No. 84 g5912, Affinity Biosciences), and horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (Batch No. F300405, Abways Technology) were used in this study.

The HK-2 cells were lysed in immunoprecipitation lysis buffer with protease inhibitor (bioswamp, Cat. No. PAB180006). Then the cellular lysate supernatant was obtained by centrifugation (10,000 × g, 30 min, 4℃). Later the protein concentration was assayed by the BCA kit (bioswamp, Cat. No. PAB180007). For immunoprecipitation of mindin, the cell samples containing 500 μg of total protein were pre-cleaned via incubition with protein A/G magnetic beads (MCE, Cat. No. HY-K0204) for 30 min at 4℃. The supernatant was incubated with anti-mindin mouse monocloning antibody (1:1000, Santa Cruz) at 4 ℃ overnight. The acquired immune complexes were precipitated by protein A/G agarose beads at 4 ℃ for 2 h. The immune-precipitates were washed 5 × with lysis buffer on ice and re-suspended with Laemmli sample buffer. Then the samples were analysed by SDS-PAGE and immunodetected using anti-TLR4 antibody (1:1000, Proteintech Group, Inc, China, 66350-1-Ig), and checked by a mouse anti-mindin antibody(Santa Cruz, USA, sc-166868). Furthermore, the whole cell lysates were performed to measure mindin and TLR4 proteins by immunoblotting.