To extract membrane proteins from cells, cell lysates were prepared according to the manufacturer’s protocol using the Mem-PER™ plus membrane protein extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) and processed.
Each sample (40 μg) was electrophoresed on 10% polyacrylamide gels (Mini-PROTEAN TGX precast gels; Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene chloride membranes (Millipore, Bedford, MA, USA). After blocking with 3% nonfat milk for 1 h at 25 °C, the membranes were incubated overnight at 4 °C with primary antibodies against apolipoprotein D/APOD (1:100, 347-MSM4-P1; ThermoFisher Scientific), CAV1 (1:1200, sc-53564; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (1:2000; Santa Cruz Biotechnology) in a blocking solution. The next day, the samples were incubated with goat anti-mouse IgG H & L (Horseradish peroxidase) (ab205719; abcam, Cambridge, UK) at 1:1000 dilution for 2 h at 37 °C. After washing, immunoreactive protein bands were visualized using an electrochemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). Images of the bands were acquired using a chemiluminescence imager (ImageQuant LAS4000mini; GE Healthcare, Chicago, IL, USA). Image analysis was performed using ImageJ (ver. 1.53p, National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times.
Each sample (40 μg) was electrophoresed on 10% polyacrylamide gels (Mini-PROTEAN TGX precast gels; Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene chloride membranes (Millipore, Bedford, MA, USA). After blocking with 3% nonfat milk for 1 h at 25 °C, the membranes were incubated overnight at 4 °C with primary antibodies against apolipoprotein D/APOD (1:100, 347-MSM4-P1; ThermoFisher Scientific), CAV1 (1:1200, sc-53564; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (1:2000; Santa Cruz Biotechnology) in a blocking solution. The next day, the samples were incubated with goat anti-mouse IgG H & L (Horseradish peroxidase) (ab205719; abcam, Cambridge, UK) at 1:1000 dilution for 2 h at 37 °C. After washing, immunoreactive protein bands were visualized using an electrochemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). Images of the bands were acquired using a chemiluminescence imager (ImageQuant LAS4000mini; GE Healthcare, Chicago, IL, USA). Image analysis was performed using ImageJ (ver. 1.53p, National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times.