Bca protein assay kit

This assay was performed as previously described^{56 (link)}. KGN cells were seeded in 24-well plates and left overnight. On the following day, the medium was replaced with serum-free medium and the cells were pretreated with the test chemicals for 24 h. Testosterone (10 nM) was then added to each well, and cells were incubated for a further 24 h. The culture medium and cell lysate were collected and stored at −20 °C. The levels of 17β-estradiol in the culture medium were quantified by use of a magnetic particle-based 17β-estradiol enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Bio-Ekon Biotechnology, Beijing, China). The intensities were measured at 550 nm with a Verioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA) and normalized to the total cellular protein content. Protein determination was conducted with a bicinchoninic acid (BCA) protein assay kit (Bestbio, Shanghai, China).

Tobacco leaves that transiently expressed 35S::MdCPK1a-GFP and 35S::GFP (control) were homogenized in liquid nitrogen. The nuclear proteins, cytoplasmic proteins and plasma membrane(PM) proteins were extracted with the Plant Nuclear, Cytoplasmic and Membrane Proteins Extraction Kit (BestBio, Shanghai, China) from plant tissues, respectively. Total proteins were extracted with extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1%SDS, 1%Triton X-100), and followed by centrifugation at 12000 rpm for 15min, and the supernatants were collected.
Following standardization of protein concentrations using BCA Protein Assay kit (BestBio, Shanghai, China), Equal amounts of protein were employed in 10% SDS-PAGE and transferred to the NC membrane. After blocking with 5% skimmed milk powder in PBST (0.5% Tween in PBS) at room temperature for 2 h, the membrane was incubated with Anti-GFP rabbit polyclonal antibody (Sangon Biotech, Shanghai, China) at 4°C overnight. After this, the membrane was rinsed three times with PBST for 5 min and then incubated with the HRP-conjugated Goat Anti-Rabbit IgG (Sangon Biotech, Shanghai, China) for 1 h. Subsequently, the membrane was washed with PBST, visualized by enhanced chemiluminescence and then detected in the Tanon 2500 chemiluminescence imaging system (Shanghai, China).

The cells were placed in cold RIPA solution (Solarbio, C0065) with PMSF (Solarbio, P0100) after washing the cells twice with phosphate buffer. Subsequently, they were pyrolysed with a protease inhibitor mixture for 30 min. The supernatant was centrifuged and harvested to analyse the protein concentration using the BCA Protein Assay Kit (BestBio, A045-4). Immunoblotting was conducted using the SDS-PAGE electrophoresis system. Briefly, 25 μL of sample was loaded and electrophoresed on a 10% SDS reducing gel. Blots were blotted onto a polyvinylidene fluoride membrane and incubated with primary antibodies against TLR3 (1 : 1000, Abcam, ab13915, USA), TLR4 (1 : 500, Abcam, ab13556), TLR6 (1 : 1000, Proteintech, 22240-1-AP), IDO1 (1 : 500, Abcam, ab55305), and NF-κB (1 : 500, Abcam, ab14059) overnight at 4°C. On the following day, the blot was washed three times with TBST and incubated with horseradish peroxidase-conjugated (HRP-conjugated) AffiniPure Goat anti-Mouse IgG (H+L) (1 : 5000, Proteintech, SA00001-1, USA) or HRP-conjugated AffiniPure Goat anti-Rabbit IgG (H+L) (1 : 5000, Proteintech, SA00001-2). Further, the blot was kept at 25°C for 1 h in TBST. Finally, proteins on the washed membrane were visualised using the TBST (Solarbio, T1082).

Colon tissues of all groups were homogenized using RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 5 mM EDTA, 1 mM Na₃VO₄] with protease inhibitor (1:100) on ice. The nuclear and cytoplasmic proteins were extracted using available nuclear and cytoplasmic protein extraction kits (Beyotime biotechnology, China) in accordance with the manufacturer's instructions. The concentration of protein was analyzed using the Bestbio BCA Protein Assay Kit (Shanghai, China). Protein lysates (30 μg) were separated by polyacrylamide gel-electrophoresis, and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Whereafter, the membranes were blocked with 5% skim milk in TBST for 1 h and washed with TBST for three times. The membranes were incubated overnight at 4 °C with primary antibodies (Nrf2 or Keap1, diluted 1:2,000). The membranes were washed three times, and incubated with secondary antibodies (diluted 1:2,000) for 1 h. Histone H3 and β-actin served as the loading control, respectively. The chemiluminescence signals obtained were quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).

SMSCs were seeded in 6-well plates and transfected with different treatment groups. Total proteins were extracted using lysis buffer and the concentration was measured by a bicinchoninic acid (BCA) protein assay kit (BestBio, Shanghai, China). Subsequently, proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PDVF) membranes (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with Quickblock closed solution (Beyotime, Shanghai, China) for 1 h at room temperature and incubated overnight at 4 °C, with primary antibodies specific for anti-MYHC (Santa Cruz Biotechnology, Dallas, TX, USA, 1:1000), anti-caspase3 (Abcam, Cambridge, UK, 1:1000), anti-HDAC4 (Zenbio, Chengdu, China, 1:1000) or anti-β-tubulin (Zenbio, Chengdu, China, 1:1000). Then, the membranes were washed three times with washing buffer (Beyotime, Shanghai, China). Next, the PDVF membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:1000). Finally, protein bands were detected using the chemiluminescence (ECL) system (Beyotime, Shanghai, China) and analysis was done using the ImageJ software (National Health Institute, Bethesda, MD, USA). β-tubulin serves as the internal control of the experiment and each treatment group had three independent replicates.

The activities of Caspase 3 and 9 in MDA-MB-231 cells were assessed based on the specific protease-peptide substrate chromogenic reaction. In brief, MDA-MB-231 cells cultured in 6-well plates at 4 × 10⁵ cell/well were treated for 12 h with PBS (as control) and FCP (5 μM) or FCP/HA ([FCP] = 5 μM). After that, the cells were harvested, lysed, and centrifugated. Then, aliquots of supernatants were collected and incubated with the peptide substrates of Caspase 3 (Ac-DEVD-pNA) and 9 (Ac-LEHD-pNA) (Enzo Life Sciences, Inc., USA), respectively. The activities of Caspase 3 and 9 were determined based on the absorbance at 405 nm by Thermo Scientific Varioskan Flash multimode reader. The total protein content of each sample was determined to normalize the obtained values by a BCA protein assay kit (Bestbio, China), and the activity ratio was calculated as compared to the blank control.