Bca protein assay kit

Protein from liver samples was extracted by RIPA Lysis Buffer (P0013, beyotime, Shanghai, China) containing 1 mmol/L protease inhibitor (ST506, PMSF, beyotime, Shanghai, China) and quantified with a BCA protein assay kit (KGP902, keygentec, Nanjing, China) according to kit instructions. Proteins were separated on SDS-PAGE and then electrophoretically transferred onto PVDF membrane (Millipore, Code No. IPVH00010, Burlington, MA, USA). Membranes were blocked for 2 h in TBST containing 5% nonfat dry milk at room temperature. Then, membranes were incubated overnight at 4 °C in antibody dilution buffer containing primary antibodies (details are shown in Table 2). A goat anti-rabbit IgG (H + L) secondary antibody (Bioker biotechnology, code BK-M050, Hangzhou, China) with 1/20,000 dilution was used in the detection of specific proteins. GAPDH was used as control. Finally, the signals were detected by adding ECL Star Chemiluminescence solution (P0018, Beyotime Biotechnology, Shanghai, China). Band intensities were determined by using Image J software. The relative expression of target proteins = the optical density of target proteins/the optical density of GAPDH.

Frozen colonic tissues were homogenised with lysis buffer to extract the total protein. The homogenate was centrifuged at 12,000 ×g at 4 °C for 5 min. The supernatant obtained was used for the quantification of the levels of TNF-α, IL-1β and IL-6 using commercially available ELISA kits (TNF-α, IL-1β and IL-6, Ray Biotech, Inc.) according to the manufacturer’s instruction. The amount of total protein concentration in the supernatant was determined using the BCA protein assay kit (Jiangsu KeyGen Biotech Co., Ltd., Nanjing, China).

Fresh mycelia (200 mg) of each culture treated with DMSO or different concentrations of Pro enantiomers were harvested, finely ground and suspended in 1 mL of extraction buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonylfluoride (PMSF), pH 7.5) containing 10 μL of protease inhibitor cocktail (Sangon Co., Shanghai, China) and homogenized with a vortex shaker. Then the supernatants were collected after the lysate was centrifuged at 10,000× g for 20 min at 4 °C. The protein concentration was determined using the BCA protein assay kit (Keygen, Nanjing, China). Equal amounts of protein (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filters (PVDF, BioRad, Hercules, CA, U.S.A). The CYP51 protein was detected using the CYP51A1 antibody (1:1000, Abcam, Cambridge, MA, USA) overnight at 4 °C after 1 h blocking with 5% non-fat milk in TBST and then with secondary antibody (1:5000) for 2 h at room temperature. Blots were developed with the ECL system (GE Healthcare). Equal loading of protein is normalized by β-actin as an internal standard. Band intensity was quantified by densitometry using Image J program (Image Processing and Analysis in Java; NIH, Bethesda, MD, USA).

CRC-derived conditioned medium was obtained from cells cultured in RPMI-1640 medium added with 10% exosome-depleted FBS (ABW, Gaithersburg, MD, USA) for 48 h, and remove cells and debris by centrifugation. Exosomes were purified from serum of CRC patients or conditioned medium by ultra-centrifugation and resuspended in PBS^{6 (link)}. The particle sizes of exosomes were measured by ZetaView (Particle Metrix, Germany). Exosomes were observed on transmission electron microscope (Hitachi H-7500, Japan). For quantification of exosomes, the amount of exosomes was measured via BCA Protein Assay kit (KeyGEN BioTECH, Nanjing, China). For cell treatment, 2 × 10⁵ receptor cells were added with 2 µg exosomes for 48 h.

The total and nuclear protein were, respectively, extracted from the aforementioned K1 and IHH4 cells as required. The protein concentration was quantified by the BCA Protein Assay Kit (KeyGEN BioTECH). Equal amount of protein was separated by 10% or 12% SDS‐PAGE and transferred onto PVDF membranes (Millipore) that were subsequently blocked by 5% bovine serum albumin (BSA) for 2 hours at room temperature. Then, the PVDF membranes were incubated with respective primary antibodies at 4°C overnight and horseradish peroxidase (HRP) labelled secondary antibody for an additional 2 hours at 37°C. Finally, chemiluminescence (ECL) kit (Millipore) was used to visualize antibody activity that was detected by Molecular Imager Gel Doc XR System (Bio‐Rad). The mean intensity of protein bands was quantified with Image‐Pro Plus 6. The primary antibodies were as follows: rabbit anti‐VDR, anti‐PTPN2, anti‐p‐STAT3, anti‐STAT3, anti‐cleaved caspase‐3, anti‐Bcl‐xl (Abcam), anti‐GAPDH and anti‐PCNA (Cell Signaling Technology).

The homogenate was made from the brain tissue obtained after reperfusion, which was further centrifuged (13200×g, 20 min, 4°C). The protein contained in supernatant was evaluated by BCA protein assay kit, standardized by bovine serum albumin (KeyGEN, Nanjing, China). The nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, U.S.A.) was exerted to receive the samples (50 μg each) separately, followed by blockage of nonfat milk for 2 h before the respective overnight incubation with anti-phospho-p53 (Ser¹⁵) antibody (Cell Signaling Technology, Woburn, MA, U.S.A.) (1/1000 diluted), PUMA (Cell Signaling Technology),(1/1000 diluted), DRAM (Assay Designs and Stressgen Bioreagents, Ann Arbor, MI, U.S.A.) (1/1000 diluted), cathepsin B (Santa Cruz) (1/1000 diluted), cathepsin D (Santa Cruz) (1/1000 diluted), Bax (Santa Cruz, CA, U.S.A.) (1/1000 diluted), Bcl-2 (Cell Signaling Technology) (1/1000 diluted), LC3 (Abcam, Cambridge, MA, U.S.A.) (1/1000 diluted) and Beclin 1 (Cell Signaling Technology) (1/1000 diluted). They were also administrated peroxidase-labeled goat anti-rabbit IgG (Santa Cruz). Their blots were revealed by the chemiluminescence substrate (ECL Plus) before intensity evaluation. The total proteins were exerted for comparison to evaluate the corresponding targeted proteins’ phosphorylation levels while GAPDH protein was exerted for loading controlling.