X ray imaging system

Protein samples from the supernatant of hepatic tissue were electrophoretically separated via SDS‒PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, USA). After blocking with bovine serum albumin for 90 min at room temperature, the membranes were incubated overnight at 4 °C with the following antibodies: anti-CYP7A1 (bs-21430R, 1:1000, Bioss, Beijing, China), anti-HMGCR (ab174830, 1:5000, Abcam), anti-Leptin (bs-0409R, 1:1000, Bioss), anti-OB-Rb (ab5593, 1:2000, Abcam), anti-JAK2 (ab108596, 1:5000, Abcam), anti-phosphorylated (p)-JAK2 (ab32101, 1:5000, Abcam), anti-STAT3 (ab68153, 1:2000, Abcam), anti-p-STAT3 (ab76315, 1:1000, Abcam), and anti-GAPDH (ab181602, 1:10,000, Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (bs-40295G-HRP, 1:10,000, Bioss) at room temperature for 1 h. An ECL kit (Solarbio, Beijing, China) was used to visualize the protein bands. The blots were imaged using an X-ray imaging system (Bio-Rad, USA). GAPDH was used as the internal reference.

H1975 with different treatments were cultured in the 2D/3D cell culture system for 48 h and then lysed with RIPA lysis (Beyotime, Haimen, China) buffer at 4 °C. The DNA or Matrigel was removed via centrifugation (ThermoFisher, Waltham, MA, USA) at 10,000 rpm for 15 min. Total protein was quantified with a BCA Protein Assay Kit (ThermoFisher, USA) and then mixed with loading buffer and boiled at 100 °C for 10 min. Protein extracts (30 μg) were resolved and separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membrane was incubated in the sequence of blocking solution (5% non-fat milk) for 1 h, primary antibody EGFR, β-Actin, p62, and LC3b solution for 3 h, and HRP-tagged secondary antibody solution for 1 h at room temperature with gentle rocking. The nitrocellulose membranes were washed three times with phosphate-buffered saline with 0.05% Tween-20 (PBST) buffer before every incubation. Finally, the membrane was incubated with enhanced chemiluminescence for 5 min, and the blot was imaged using an X-ray imaging system (Bio-Rad, Hercules, CA, USA). Every experiment was performed at least 3 times. The relative ratio of EGFR, p62, and LC3b-II to β-Actin was evaluated using Image J software.