Bicinchoninic acid protein assay kit

Freshly isolated liver tissue and BRL 3A cells were lysed; the protein concentration was determined using a bicinchoninic acid protein assay kit (Vazyme, Nanjing, China). Equivalent amounts of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Danvers, MA, USA). Each membrane was blocked for 2 h using 5% skim milk, incubated overnight with primary antibodies at 4 °C, and then with secondary antibodies for 2 h at room temperature. The primary antibodies were those against: MyD88, GAPDH, TRAF6, p-TAK, IκBα, p-IκBα, NFκB, p-NFκB, TLR2, and NOS2. The membranes were imaged with chemiluminescence. Data quantification analyses were performed using Image J (NIH Image, Stuttgart, Germany).

Chinese motherwort (L. artemisia) (Scientific Chinese Medicine) was obtained from the Chiayi Chang Gung Memorial Hospital. Roswell Park Memorial Institute (RPMI)-1640 basal medium supplemented with fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NE, USA). A cell counting kit-8 (CCK-8) was obtained from Dojindo (Kumamoto Prefecture, Japan). The wound healing assay, annexin V, fluorescein isothiocyanate, propidium iodide, cell cycle and apoptosis analysis kit, and lysis buffer for the radioimmunoprecipitation assay were obtained from Blossom Biotechnologies (Taipei, Taiwan). Phosphatase inhibitor cocktail tablets (PhosSTOP) were received from Roche (Basel, Switzerland). Bovine serum albumin fraction V and ultrasensitive enhanced chemiluminescence substrate were obtained from Biosharp (Hefei, China). A bicinchoninic acid protein assay kit was obtained from Vazyme (Nanjing, China). Antibodies against cleaved caspase-3, PARP, cleaved PARP, Bax, Bcl-2, p-p38, p-JNK, p-ERK, and GAPDH and horseradish peroxidase-conjugated goat antirabbit antibodies were purchased from CST (Boston, MA, USA). All other chemicals were of analytical grade.

H9 cells were harvested and lysed in lysis buffer. Protein concentrations were measured using the bicinchoninic acid protein assay kit (Vazyme, Nanjing, China), followed by heating under reducing conditions. A total of 30 µg of protein was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and subsequently transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the following antibodies: GAPDH (1:10,000), lamin B (1:1,000), PD-1 (1:1,000), CTLA-4 (1:200), NFATc1 (1:1,000), and p-NFATc1 (1:10,000).