Bca test kit

Total protein from THCA cells was extracted with the help of RIPA lysis buffer (Thermo Fisher Scientific) having protease inhibitor cocktail (Roche, Basel, Switzerland). The quantity of individual protein samples was evaluated availing a BCA test kit (Thermo Fisher Scientific). Through a 12 % SDS-PAGE, protein samples (20 μg) were separated at 150 V for 1.5 h and then transferred onto PVFD membranes (Millipore). Following blocking these with 5 % non-fat milk, the membranes were treated with primary antibodies at 4 °C overnight. The following antibodies were used in this study at a dilution of 1:1000 and were acquired from abcam, Cambridge, UK - Bcl-2 (ab32124), Bax (ab32503), EVA1A (ab216043), and GAPDH (ab9485). The membranes were then treated with hrp-conjugated goat anti-rabbit IgG secondary antibody (abcam, 1:10,000). Finally, each membrane was visualized using a chemical immunogenicity system (GE Healthcare, Chicago, IL, USA), and ImageJ software (NIH, Bethesda, MD, USA) was employed for quantitation of the intensities of the protein bands.

To assess the effects of compounds on IκBα protein expression, cells were treated with the substances for 30 min before exposure to LPS (1 μg/mL) for one hour.^{40 (link)} Also, to investigate the expression of STAT3, p-STAT3, BAX, and Bcl-2 proteins, cells received IL-6 (50 ng/mL) for four hours after treatment with compounds.^{41 (link)} The supernatant of centrifuged lysates was used for western blot analysis. The protein content was evaluated using a BCA test kit (Thermo Fisher Scientific, Oxford, UK). The samples were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, St. Louis Missouri, USA) after loading on 10% SDS-PAGE gels. After membrane blocking, the membranes were coated with the primary antibodies at 1:1000 dilution against BAX, Bcl-2, IκBα, STAT3, p-STAT3, and β-actin (Sigma-Aldrich, Munich, Germany) overnight at 4 °C. Afterward, the membranes were then coated with HRP-conjugated anti-mouse immunoglobulin G secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA). Eventually, enhanced chemiluminescence (ECL) (SuperSignal, Thermo Fisher Scientific, Oxford, UK) was used for detection.