Brefeldin A 是一种布雷菲尔德青霉代谢物，它是一种大环内酯，具有广泛的抗生素活性。 它还是 HCT 116 细胞中蛋白质转运的 ATPase 抑制剂（IC50=0.2 μM），诱导 Y 细胞分化和凋亡。

Brefeldin A , a Penicillium brefeldianum metabolite, which is a macrocyclic lactone exhibiting a wide range of antibiotic activity. It is also an ATPase inhibitor for protein transport in HCT 116 cells(IC50=0.2 μM), inducing Y cell differentiation and apoptosis.

在HF4.9 和 HF28RA 细胞中, Brefeldin A（25 ng/mL）完全抑制细胞生长。在HF1A3 细胞中,Brefeldin A (75 ng/mL) 完全抑制细胞生长。

ELISA-based active site binding assay: Samples (lysed cells or tissue homogenates) are treated for 1 h at room temperature with the biotinylated active site probe PR-584 (5-15 μM). Samples are denatured by addition of SDS (0.9% final) and heating to 100 °C for 5 min. The denatured samples are transferred to a 96-well or 384-well filter plat, mixed with streptavidin-sepharose beads (2.5-5 μL packed beads/well), and incubated for 1 h at room temperature on a plate shaker. The beads are washed 5 times with 100-200 μL /well of ELISA buffer (PBS, 1% bovine serum albumin, 0.1% Tween-20) by vacuum filtration. The beads are incubated overnight at 4 °C on a plate shaker with the following antibodies recognizing the six catalytic subunits diluted into ELISA buffer: β5, β1, and β2 diluted 1:3000, LMP7 and LMP2 diluted 1:5000, and MECL-1 diluted 1:1000. The beads are washed 5 times with 100-200 μL /well of ELISA buffer and incubated with HRP-conjugated secondary antibody diluted 1:5000 in ELISA buffer and incubated 2 h at room temperature on a plate shaker. The beads are washed 5 times with 100-200 μL /well of ELISA buffer and developed for chemiluminsecence signal using the supersignal ELISA pico substrate following the manufacturer's instructions. Luminescence is measured on a plate reader and converted to ng of proteasome or μg/ml of lysate by comparison with 20S proteasome or untreated cell lysate standard curves. For proteasome inhibitor studies, active site probe binding values are expressed as the percent of binding relative to DMSO treated cells.

HF1A3, HF4.9 cell viability upon the treatments is tested using double staining of cells with YO-PRO 1/PI and SYTO16/PI probes. To access cell proliferation, cells are treated with 0–100 ng/mL Brefeldin A in complete medium for 20 hours before adding 1 μCi/mL [methyl-3H]-thymidine for additional 4 hours at 37 °C. The incorporated radioactive thymidine is quantified by scintillation counting with Microbeta counter. To examine long-term effects of Brefeldin A treatment, cells are seeded at initial concentration 105 cells/mL and treated with 0-75 ng/mL Brefeldin A for up to 5 days. At the time indicated, a sample of cells is removed and viable cell number is assessed by standard Trypan Blue exclusion assay.(Only for Reference)

20350-15-6

C16H24O4

280.364

Cyanein;Decumbin;Ascotoxin;BFA;布雷非德菌素 A

Ethanol:2.8 mg/mL (10 mM)
DMSO:14 mg/mL (50 mM)
Powder: -20°C for 3 years
In solvent: -80°C for 2 years