Anti bid

BE(2)-C cells were seeded in 48-well plates and labeled with the following primary antibodies: anti-HIF-1α (Abcam, 1:500), anti-noxa (Abcam, 1:500) and anti-bid (Abcam, 1:500). Primary antibodies were labeled using fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies (Jackson, 1:1000). The cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China).

The protein extracted from hippocampus or primary neuron homogenates was used for western blotting. The membranes were incubated in primary antibodies including anti-RARα (1:250, Abcam, USA), anti-β-actin (1:150, Santa Cruz, USA), anti-PI3K (1:200, Abcam, USA), anti-p-Akt (1:250, Abcam, USA), anti-Akt(1:250, Cell Signaling, USA), anti-p-Bad(1:100, Santa Cruz, USA), anti-Bad (1:200, Cell Signaling, USA), anti-caspase-3(1:100, Santa Cruz, USA), anti-caspase-8(1:100, Santa Cruz, USA), anti-Bcl-2 (1:150, Santa Cruz, USA), anti-Bax(1:100, Santa Cruz, USA), and anti-Bid(1:200, Abcam, USA) at 4 °C overnight, respectively. The blot was probed with an enzyme-linked secondary antibody (typically horseradish peroxidase) for 1h at room temperature. Then, the blot was stained with 3,3′-diaminobenzidine (DAB) (Tiangen, China) and the signals from the chemiluminescent detection reagents were photographed using an ECL Imaging System (BioRad, USA). The samples were normalized to β-actin.