Goat anti mouse immunoglobulin g igg

Cells were lysed with 200 μL lysis buffer containing 20 mmol/L HEPES, 25 mmol/L MgCl, 5 mmol/L KCL, 0.5% (v/v) complete protease inhibitor, and Triton X-100. Then, the debris was removed by centrifugation at 12,000× g at 4 °C for 10 min. Equal amounts of cell protein (typically 80 µg) were separated using 8% precast SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA) and electrophoretically transferred to PVDF membranes (Millipore, Beijing, China). The membranes were subsequently probed individually with the following polyclonal primary antibodies: TCF2 antibody (1:500, ProSci Inc., Poway, CA, USA); GLUT2 antibody, PDX1 antibody, GCLc antibody, GCLm antibody, pAKT antibody, AKT antibody, pERK antibody, and ERK antibody (1:500, BD Transduction Laboratories, San Jose, CA, USA). Detection was performed by incubation with goat anti-mouse immunoglobulin G (IgG; 1:5000, Sigma, St. Louis, MO, USA) followed by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Ltd., Piscataway, NJ, USA). The intensity of the bands was measured using an image analysis system with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA).

RAW 246.7 cells were lysed with 200 μL lysis buffer containing 20 mmol/L HEPES, 25 mmol/L MgCl, 5 mmol/L KCL, 0.5% (v/v) complete protease inhibitor, and Triton X-100. Then, the debris was removed by centrifugation at 12,000× g at 4 °C for 10 min. Equal amounts of cell protein (typically 80 μg) were separated using 8% precast SDS-PAGE gels (Invitrogen) and electrophoretically transferred to PVDF membrane. The membranes were subsequently probed individually with 1:150 polyclonal primary ABCA1 antibody, ABCG1 antibody, SR-A antibody, CD36 antibody, or BMPR-2 antibody (BD Transduction Laboratories, San Jose, CA, USA) or 1:1000 SMAD1/5/8 antibody (Cell Signaling Technology, Beverly, MA, USA). Detection was by incubation with goat anti-mouse immunoglobulin G (IgG; 1:5000; Sigma) followed by enhanced chemiluminescence (ECL, Amersham Pharmacia, NJ, USA). The intensity of the bands was measured using labwords analysis software (Shenteng, Shanghai, China).

Anti-β-actin (Millipore, Temecula, CA; MAB1501), anti-UCP1 (Abcam, Cambridge, MA, USA; ab10983), anti-phospho (T172) AMPKα (Abcam, Cambridge, MA, USA; ab2535), anti-AMPKα (Abcam, Cambridge, MA, USA; ab2532S), anti-Prdm16 (Abcam, Cambridge, MA, USA; ab106410), goat anti-mouse immunoglobulin G (IgG) (Sigma, St. Louis, MO, USA; #AP124P), goat anti-rabbit (Sigma, St. Louis, MO, USA; #401353), anti-4,6-diamidino-2-phenylindole (Sigma, St. Louis, MO, USA), and anti-rabbit antibody Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA; A11012) were used in this study. Rg3 was purchased from Abcam (Abcam, Cambridge, MA, USA; ab141938).