Complete lysis m buffer solution

Total protein from cell lines and PDCs were lysed in cOmplete Lysis-M buffer solution (Roche, Basel, Switzerland), and protein concentrations were determined using a Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (30 μg) were boiled for 5 min at 90°C and then separated in a 4–12% Bis-Tris gel (Invitrogen) utilizing the Invitrogen Novex gel running apparatus at 110 V for 90 min in MOPS running buffer. Proteins were transferred to a nitrocellulose membrane (Whatman, Maidstone, UK) at 250 mA for 2 h in Transfer buffer (Biosaesang, Seongnam, South Korea) on ice. The membranes were blocked with 5% skim milk in TBS buffer containing 0.1% Tween 20 and incubated overnight at 4°C with specific primary antibodies. The antibodies were anti-HER2 (phospho Tyr1248, 1:1,000, Cell Signaling Technology (CST), Danvers, MA, USA), anti-HER2 (D8F12, 1:1000, CST), anti-cyclin E1 (D7T3U, 1:1,000, CST), anti-cyclin E1 (phosphor Thr62, 1:1,000, CST), and β-actin (C4, 1:3,000, Santa Cruz Biotechnology, Dallas, TX, USA). Horseradish peroxidase-conjugated anti-rabbit or mouse IgG (Bio-Rad) was used as secondary antibody. Signals were detected by chemiluminescence using ECL Western Blotting Substrate (Thermo Fisher Scientific) and visualized using an LAS-4000 (Fujifilm, Tokyo, Japan).

Total cell lysates from the A549 lung cancer cell lines were prepared using the Complete™ Lysis-M buffer solution (Roche Life Science, Penzberg, Germany). Protein extracts were resolved using 4–20% Mini-PROTEAN TGX™ Precast Protein Gels (Bio-Rad, Berkeley, CA, USA) and transferred onto iBlot® PVDF gel transfer stack membranes (Thermo Fisher Scientific, Waltham, MA, USA). After blocking non-specific binding sites for 1 h in 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the membranes were incubated overnight at 4 °C with specific primary antibodies. The antibodies included anti-p-EGFR (phospho Y1092) antibody (1:1000, Abcam, Cambridge, UK) and anti-beta actin (1:2000, Abcam, Cambridge, UK). These were used following the manufacturers’ instructions.

To analyze changes in the expression level of ADH after exposing HepG2 cells to no compound (control), 100 μM BPA, and a mixture of 100 μM BPA and 2.5 mg/mL pear extract, we performed Western blot analysis. Total HepG2 cell lysates were prepared with cOmplete™ Lysis-M buffer solution (Roche Life Science, Penzberg, Germany). Protein extracts were resolved by 4–20% Mini-PROTEAN TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred onto iBlot^® PVDF gel Transfer Stacks (Thermo Fisher Scientific Korea). After blocking non-specific binding sites for 1 h in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TBST), each membrane was incubated overnight at 4 °C with specific primary antibodies. Following manufacturer’s instructions, we used ADH antibody (1:1000, Abcam, Cambridge, UK) and anti-beta actin (1:2000, Abcam). The membranes were washed in TBST and incubated further with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (1:5000, Abcam) at room temperature. Protein bands were detected using an enhanced ECL^™ Western blotting detection kit (GE Healthcare, Little Chalfont, UK).