Phospho erk tyr204

Whole-cell lysates were prepared in a cell lysis buffer (Cell Signaling Technology) containing a protease (Tech & Innovation^TM, Bucheon, Korea) and phosphatase (sc-45065; Santa Cruz Biotechnology, Dallas, TX, USA) inhibitor cocktail. Proteins were separated on 10% or 12% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes using an iBlot^TM dry blotting system (Invitrogen), and analyzed using primary antibodies against the following proteins: SHP2 (sc-280; Santa Cruz), ERK (sc-154; Santa Cruz), phospho-ERK (Tyr204) (sc-7383; Santa Cruz), HA (H6908; Sigma-Aldrich), Gab1 (06-579; Millipore, Billerica, MA, USA), GAP (06-157; Upstate Biotechnology, Charlottesville, VA, USA), EGFR (2232; Cell Signaling Technology), STAT3 (9132; Cell Signaling), phospho-STAT3 (Tyr705) (9131; Cell Signaling), and β-actin (A5441; Sigma-Aldrich) or vinculin (v9131; Sigma-Aldrich). Blots were developed using the SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA).

Antibody against LDLR (Cat # ab-30532),  MCT-1 (Cat # ab-90582), MCT-3 (Cat # ab-60333) and SR-B1 (Cat # 217318) were purchased from Abcam (Cambridge, UK); antibodies against FASN (Cat # sc-55580), MCT-4 (Cat # sc-50329), MCT-2 (Cat # sc-166925), LDHA (Cat # sc-137244), LXRα/β (Cat # sc-377260), ACAT-1 (Cat # sc-517387), ACAT-2 (Cat # sc-293307), phospho-ERK (Tyr 204) (Cat # sc-7383), ERK (Cat # sc-154), Cyclin A (Cat # sc-751), CDK 2 (Cat # sc-163), Cyclin E (Cat # sc-198), β-tubulin (Cat # sc-9104), GAPDH (Cat # sc-20357), β-actin (Cat # sc-1615), and HRP conjugated secondary antibodies: anti-rabbit (Cat # sc-2030), anti-mouse (Cat # sc-2031), anti-goat (Cat # sc-2033) were purchased from Santa Cruz Biotechnology (CA, USA). Human LDL cholesterol (Cat # 360–10) and human HDL cholesterol (Cat # 361–10) were purchased from Lee Biosolution (MO, USA); Water soluble-free cholesterol and Dextran sodium sulfate (DSS) (Cat # 160110) were purchased from MP Biomedicals (CA, USA). Azoxymethane (AOM) (Cat # A5486), 2-Deoxy-D-glucose (2-DG) (Cat # D8375) and Oxamate (Cat # O2751) were purchased from Sigma Aldrich (MO, USA).

Western blotting was performed to determine the expression of EGFR signaling pathway-related proteins in lung cancer cells treated with candidate compounds, as described previously [60 (link)]. H1975 cells were seeded in culture medium and drug solution for 48 h. A549 cells were pretreated with candidate compounds at desired concentrations for 48 h before exposure or non-exposure to 20 ng/mL EGF for 30 min. The cells were then lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% NP-40. The cell lysates were subjected to Western blotting. Recombinant human EGF was purchased from Cascade Biologics (Portland, OR, USA). Antibodies against phospho-EGFR (Tyr1068), phospho-PI3K (Tyr199/Tyr458), phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), and AKT were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against EGFR, PI3K, STAT3, ERK, GAPDH, and phospho-ERK (Tyr204) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phospho-AKT (Ser473) were purchased from Millipore (Billerica, MA, USA). Antibodies against Src were produced in our laboratory (ATCC CRL-2651). Antibodies against phospho-Src (Tyr418) were purchased from Invitrogen (Carlsbad, CA, USA). GAPDH was used as the loading control. Protein expression was quantified with ImageJ software.