Anti bad

Tissues and cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). The protein contents were determined by a BCA protein detection kit (Beyotime, Shanghai, China). The proteins were separated by electrophoresis on sodium dodecyl sulfate–polyacrylamide (SDS PAGE) gels and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 2 h at room temperature, and then probed with the indicated primary antibodies overnight at 4 °C with gentle shaking. After washing with phosphate buffered saline with Tween 20 (PBST), the membrane was developed with a secondary antibody (HRP-conjugated goat anti-rabbit IgG; ab205718) for 1 h at room temperature. Subsequently, membranes were visualized using a chemiluminescence imaging system (Biolight, Guangzhou, Guangdong, China) according to the manufacturer’s protocol. The primary antibodies used were anti-CycA (ab185619, 1:1000), anti-CycD1 (ab16663, 1:100), anti-EDN1 (ab2786, 1:1000), anti-PI3K (ab278545, 1:1000), anti-CLDN1 (ab180158, 1:2000), anti-CLDN2 (ab53032, 1:500), anti-Caspase-3 (ab184787, 1:2000), anti-Bcl-2 (ab182858, 1:2000), anti-Bax (ab32503, 1:1000), anti-Bad (ab32445, 1:1000), anti-GAPDH (ab8245, 1:500) from Abcam (Cambridge, MA, USA).

The procedures were performed as previously mentioned33 and the samples were incubated with the primary antibodies mentioned blow: anti‐NeuN (1:1000, Cell Signaling Technology), anti‐Bad (1:500, Abcam) and anti‐Tuj 1 (1:1000, Neuromics). Then, on the following day, we treated the samples with secondary antibody (1:1000, Invitrogen) along with DAPI (1:800, Invitrogen).
To detect mitochondrial ROS levels, we used Mito‐SOX Red staining (Life Technologies) on the basis of the manufacturer's instructions. And we took advantage of the TUNEL assay (Invitrogen) to detect apoptosis.

Total protein was extracted from whole cells. The samples were heated at 90 °C for 10 min with the 1× sample buffer and separated by SDS-PAGE. Subsequently, the sample was electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were probed with polyclonal rabbit antibodies against anti-Bcl-2, anti-Bax, anti-cleaved-caspase-3, anti-OTUD7B, anti-TNIP2, and anti-BAD (Abcam, Cambridge, MA, USA). For loading control, the membranes were stripped and re-probed with anti-alpha-Tubulin (Abcam).