Bicinchoninic acid bca protein assay kit

Collect the correspondingly processed lung cancer cells in an EP tube, add an appropriate amount of RIPA lysate to lyse, centrifuge, and take the supernatant. Use the Bicinchoninic Acid Protein Assay kit (BCA, Solarbio, Beijing, China) to determine the total protein concentration. After adding protein buffer, bathe in boiling water for 10 min. Take an appropriate amount of total protein in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein, and then transfer the protein to a polyvinylidene fluoride membrane (PVDF) And sealed with 5% skimmed milk powder. Use the appropriate concentration of primary antibody to incubate overnight at 4 °C: anti-SPP1 (ab214050, 1:1000, Abcam), anti-COL11A1 (ab64883, 1:1000, Abcam), anti-E-cadherin, 1:1000 (3195S, Cell Signaling); anti-N-cadherin, 1:1000 (13116 T, Cell Signaling); anti-Vimentin, 1:1000 (5741S, Cell Signaling), GAPDH (ab8245, 1:1000, Abcam). Then, they were incubated with the appropriate concentration of secondary antibodies of the same species for 1 h at room temperature. After cleaning the membrane 3 times with 1XTBST, the protein bands were detected with an ECL luminescence reagent (Yuhengbio, Suzhou, China) and gel imaging analyzer (Bio-Rad, California, USA). Quantitative analysis of relative protein levels was performed with Quantity One software (Bio-Rad, California, USA).

The China Institute of Food and Drug Control provided the following reference standards: astragaloside IV, glycyrrhizic acid, calycosin-glucuronide, formononetin, ononin, calycosin-7-O-β-D- glucoside, puerarin, and digoxin (purity >98.0%). Ticlopidine (693228-63-6), α-naiflavone (604-59-1), quinidine (56-54-2), fluconazole (86386-73-4), ketoconazole (65277-42-1), and clomethiazole (533-45-9) were supplied by Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Nicotinamide adenine dinucleotide phosphate (NADPH, 704S082) and bicinchoninic acid protein assay kit (BCA, 20191123) were provided by Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Merck KGaA (Germany) provided methanol and acetonitrile (HPLC grade). All remaining chemicals were of analytical grade. HQD was self-produced by the research group (Wang et al., 2022 (link)). The content of each component in HQD is shown in Table 1.

Cells were washed twice with ice-cold phosphate-buffered saline and then disrupted using radio immunoprecipitation assay (RIPA) lysis buffer (with phosphatase and protease inhibitors). Cell lysates were centrifuged at 12,000 × g for 15 min at 4°C and the supernatant fractions were collected. Bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China) was used for the quantification of total protein. Cell lysates (20 – 40 μg) were separated by SDS-PAGE and the bands were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membranes were then blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBST) followed by overnight incubation with antibodies against KRT18 (1:1000) or GAPDH (1:2000) at 4°C. The blots were then incubated with goat anti-mouse/rabbit IgG-HRP secondary antibodies for 1 h at room temperature (RT). Following the incubation, protein bands were visualized using the enhanced chemiluminescence reagent (Millipore, Billerica, MA, United States) and Amersham Imager 600 Ultra-sensitive multi-function imager (GE Healthcare, America).