Las 1000 ccd camera

Protein samples were separated on 6% SDS-PAGE according to the method of Laemmli [16 (link)]. For immundetection, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore) using a semidry transfer unit (Hoefer TE77X semidry transfer unit). Blocking of the membrane was carried out in PBS (pH 7.4) with 5% skim milk for 1 h at room temperature or overnight at 4°C. After a washing step of 10 min in PBS (pH 7.4) with 0.05% Tween 20, membranes were incubated with mouse monoclonal anti-V5 antibody (1:5.000; Clone SV5-PK1 Acris) in PBS (pH 7.4) with 0.05% Tween 20 and 0.5% BSA. Subsequent detection occurred via peroxidase-coupled sheep anti-mouse antibody (1:5.000; GE Healthcare) and ECL Plus chemiluminescence substrate (Pierce) using a LAS-1000 CCD camera (Fuji Photo Film).

For immunoblotting of SREBP-1c, the nuclear protein extracts from each sample (10 μg/lane) were separated by 10% SDS-PAGE and blotted to a polyvinylidene difluoride transfer membrane (Amersham Hybond-P; GE Healthcare) in a semidry system. The membrane was incubated for 1 h in 5% fat-free milk, then overnight with the primary SREBP-1c antibody (2A4: sc-13551; Santa Cruz Biotechnology) diluted 1:1,000 in 5% BSA, 1× TBS, and Tween-20 and for 1 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse; Cell Signaling) diluted 1:2,000 in 5% fat-free milk, 10× TBS, and Tween-20. The membrane was washed with TBS and Tween-20 2 × 5 min and 15 min after each incubation time. Proteins were visualized using an ECL Plus kit (Amersham Bioscience) and detected in an LAS-1000 CCD camera (Fuji).

For immunoblotting of ERα, cells were lysed in Ripa buffer and proteins from each sample (10 μg/lane) were separated by 12% SDS-PAGE and blotted to a polyvinylidene difluoride transfer membrane (Amersham Hybond-P; GE Healthcare) in a semidry system. The membrane was incubated for 1 hour in 5% fat-free milk, then overnight with the diluted 1:1000 primary ERα antibody (Hc-20: sc-543; Santa Cruz Biotechnology). Bound antibodies were detected with a secondary peroxidase-conjugated anti-rabbit (anti-rabbit; Cell Signaling) diluted 1:2000 in 5% fat-free milk, 10× TBS, and Tween-20. The membranes were washed with TBS and Tween-20 2× for 5 min and 15 min, respectively, after each incubation time. Proteins were visualized using an ECL Plus kit (Amersham Bioscience) and detected in an LAS-1000 CCD camera (Fuji). Membranes were mild stripped by using a volume of buffer (15 g glycine, 1 g SDS, 10 ml Tween 20 dissolved in 1 L distilled water adjust PH to 2.2) that covered the membrane and incubated at room temperature for 10 min. The incubation was repeated and the membranes were, washed twice in TBS-T and PBS for 10 min each and re-probed with anti- β-actin monoclonal antibody (β-actin 13E5, Cell Signaling Technology) diluted 1:1000 in TBS-T, to serve as a loading control.