Polyvinylidene difluoride membranes pvdf

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14,000 × g at 4 °C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Total proteins (50 μg) were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Then the membranes were blocked with TBST buffer (NaCl 150 mmol/L, Tris 10 mmol/L, Tween-20 0.05% (v/v) pH = 7.6) containing 5% nonfat milk powder at room temperature for 2 h. The membranes were then probed overnight at 4 °C with primary antibodies, including phospho-KAP1(1:1000, Abcam, ab70369), KAP1(1:1000, Cell Signaling Technology, #4123), PCNA (1:2000, Servicebio, GB11010), Bcl-2 (1:1000, ABclonal, A0208), Bax(1:1000, ABclonal, A0207) followed by incubation with corresponding HRP-conjugated secondary antibodies (1:5000, Proteintech, SA00001-2) at room temperature for 2 h. Proteins on the membranes were visualized by adding the enhanced chemiluminescence reagent (Millipore, USA). Band intensities were analyzed using Image J 1.25 software (National Institutes of Health, Bethesda, USA) and normalized to loading controls.

HT-29 cells (6 × 10⁵) were seeded in a 10 cm dish overnight and cultured for 24 h. Then, they were treated with cisplatin (1 µM) or the mixture of compounds 6 and 7 (10, 20, and 40 µM) for 48 h. Then, the cells were harvested and centrifuged at 5000 rpm for 5 min. The harvested cells were lysed with lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 5 mM phenylmethanesulfonyluoride (PMSF), 1 mM dithiolthreitol (DTT) containing 1% Triton X-100) and centrifuged at 12,000 rpm for 15 min to removed insoluble debris. The protein content was determined using Bradford reagent (“Bio-Rad”, Hercules, CA, USA). Lysate protein (20–40 µg) was subjected to 12% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF) (“Millipore”, Burlington, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h and then incubated with the respective specific primary antibody at 4 °C overnight. Protein bands were visualized using an enhanced chemiluminescence reagent (ECL) (“Bio Rad”, Hercules, CA, USA) after hybridization with an HRP-conjugated secondary antibody. Band density was quantified using the ImageJ software.