Bca protein assay

Differentiated cells were collected in RIPA Buffer (Wako) with protease inhibitors (Roche, Basel, Switzerland), homogenized, and centrifuged. Concentration of whole cell lysate protein was determined by a BCA protein assay (TakaRa). Proteins were boiled in sample buffer (BIO-RAD) and loaded onto Mini-PROTEAN TGX Gels (BIO-RAD) separated by SDS-PAGE. Protein bands were transferred to Trans-Blot Turbo membranes (BIO-RAD), which were then immersed in TBST containing the primary antibody. Then the membranes were treated with Blocking One (Nacalai Tesque, Kyoto, Japan). After washing, the membranes were next immersed in the secondary antibody and reacted with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA). The bands were detected on LAS 4000 mini (Fuji Film, Tokyo, Japan). Western blotting was carried out with anti-HNF1A, 1:1000 (89670, CST), anti-HNF1β, 1:1000 (12533-1-AP, Proteintech) and anti-GAPDH,1:5000 (MA5-15738, Thermo Fisher Scientific). Horseradish peroxidase–conjugated second antibody (1:2000) was purchased from Santa Cruz Biotechnology (sc-2004, Santa Cruz, CA).

Cytoplasmic nucleic acid and protein released from bacterial cells treated with dehydrocorydaline were determined to evaluate the integrity of the cell membrane (Chen et al., 2020 (link)). Listeria monocytogenes cells at the exponential growth phase were harvested by centrifugation at 2500 × g for 10 min, washed with phosphate buffered saline (PBS) three times, resuspended in PBS (1 ×10⁶ CFU/mL) containing different concentrations of dehydrocorydaline (0, MIC, and MBC), and incubated at 37°C for 4 h. The cell suspension was collected at different time intervals (0, 1, 2, and 4 h) and filtered through a 0.22 μm Millipore filter. The nucleic acid released from bacterial cells was measured by UV visible spectrophotometer (Allsheng, Hangzhou, China) with optical density at 260 nm. The amount of protein leakage was quantified by BCA protein assay (Takara Bio, Mountain View, CA, United States).

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of 2D-cultured OSCC and OASC cells, SSYP, MO-1000, LEM2, and Mesimo, were measured using a Seahorse XFe96 real-time metabolic analyzer (Agilent Technologies Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. In a typical run, twenty thousand 2D-cultured cells, as described above, were placed in each well of an XFe96 Cell Culture Microplate (Agilent Technologies, #103794-100) the day before the assay and were incubated at 37 °C. On the day of the assay, the culture medium was replaced with a Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103575-100) containing 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate). The assay plates were then incubated in a CO₂-free incubator at 37 °C for 1 h prior to the measurements. OCR and ECAR were simultaneously measured in an XFe96 extracellular flux analyzer at the baseline and under the following sequential injections of 2.0 μM oligomycin, 5.0 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mixture of 1.0 μM rotenone and 1.0 μM antimycin A, and 10 mM 2-deoxyglucose. The values were normalized to the amount of protein, as determined by a BCA Protein Assay (TaKaRa BCA Protein Assay) in each well after the measurement was complete.

The maximal activities of CS, β‐HAD, and COX were measured as described elsewhere [10 (link), 17 (link)]. Frozen and crushed gastrocnemius muscles were homogenized in 100 times (vol/wt) of 100 mm potassium phosphate buffer using a μT‐01 bead crusher (TAITEC, Saitama, Japan). Maximal enzyme activity was measured spectrophotometrically. The total protein concentration in the homogenates was determined using a BCA protein assay (TaKaRa BIO Inc., Shiga, Japan), with activities normalized to total protein concentration.

Mouse heart tissue were lysed by RIPA lysis buffer (Keygen Biotech) complemented with phenylmethylsulfonyl fluoride (PMSF). BCA Protein Assay (TaKaRa) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (10–12%). Using wet tank transfer method, protein bands were transferred onto PVDF membranes (PALL). After blocked with 5% BSA and washed with TBST, protein bands were blotted with primary antibodies at 4 °C overnight as follows: Bax (Abclonal, A12009, 1:1000 dilution), Bcl₂ (affbiotech, AF6139, 1:1000 dilution), Caspase3 (Abclonal, A2156, 1:1000 dilution), COL1A2 (Bioworld, BS1530, 1:1000 dilution), NRF2 (Abclonal, A0674, 1:1000 dilution), and β-actin (Bioworld, AP0060, 1:10,000 dilution). After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (Tanon). Quantifications of Western Blots were measured with ImageJ.

Cell lysates were prepared by sonicating cells briefly in a modified RIPA buffer (0.1% SDS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate, 1% Nonidet P-40) with proteinase and phosphatase inhibitors. BCA protein assay (TaKaRa) was used for protein quantifying. The antibody used were as follows: anti-FOXP2 (Abcam, ab16046, USA), anti-p21 (Abcam, ab109520, USA), Goat anti-Rabbit, and Goat anti-Mouse infrared dye secondary antibodies (800 CW), which were purchased from LI-COR Biosciences (Lincoln, NE, USA). Proteins were visualized with Odyssey Bioanalyzer (LI-COR).