Ipvh00010

Proteins were separated on 10% or 12% SDS-PAGE gel and transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the following antibodies: SREBP1 (PA1-337, ThermoFisher), Bax (50599-2-AP, Proteintech), Bcl2 (12789-1-AP, Proteintech), Caspase 3 (19677-1-AP, Proteintech), Caspase 9 (#9502, Cell Signaling Technology), AKT (#4691, Cell Signaling Technology), p-AKT (#4060, Cell Signaling Technology), JNK (10023-1-AP, Proteintech), p-JNK (80024-1-RR, Proteintech) and β-Actin (AC004, Abconal). After washed 3 times with TBST buffer, PVDF membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific). Western blot band were visualized by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).

For western blot assay, proteins were separated on gel where appropriate. And then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next, PVDF membranes were incubated with the indicated antibodies. Lastly, PVDF membranes were incubated with anti-rabbit (Thermo Fisher Scientific, 31460) or anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).

Cells were lysed in NP40 lysis buffer and then cell lysates were centrifuged at 12,000 g, 4 °C for 20 min. 20 µL protein G agarose beads (Roche, 11243233001) were added to the supernatants for preclear. The supernatants were incubated with protein G agarose beads and indicated antibodies at 4 °C for 8 h. Then immunocomplexes were centrifuged at 4 °C for 3 min and NP-40 lysis buffer were used to wash the precipitates three times before subjected to western blot assay.
For western blot assay, proteins were separated on 10% or 12% SDS-PAGE gel where appropriate. Proteins were then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the indicated antibodies and washed 3 times with TBST (0.05% Tween-20, 150 mM NaCl and 20 mM Tris-HCl). Lastly, PVDF membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, 31460) or horseradish peroxidase-conjugated anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).