Anti bax sc 7480

Petri dishes were used to seed the cells at a density of 5 × 10⁵ cells/mL. Total proteins were extracted using the Q-proteome Mammalian Protein kit following 48 and 72 h of treatment with 0.4–2.4% of UD aqueous extract. Lowry assays were used for protein quantification. Western blot analysis was done to measure the protein expression of Bax and Bcl2. Beta-actin was used as a loading control. Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes at 0.25 mA for 75 min [30 (link)]. After blocking with 5% milk and 0.05% Tween 20 for 1 h, the membranes were incubated with the antibodies using anti-β-actin (Sc-47778), anti-Bax (Sc-7480), and anti-Bcl2 (Sc-783) overnight at 4 °C (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:2500 for anti-β-actin and 1:500 for the others. The membranes were then washed for 1 h using 1 × PBS with 0.5% Tween 20, before incubation with the secondary antibody (Promega, Madison, WI, USA) at a concentration of 1:2500 for 1 h at room temperature. After washing, the development of the membranes was done using Western blotting chemiluminescent-reagent-enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA), and a ChemiDoc XRS+ machine (BioRad, Hercules, CA, USA) was used to take images that were quantified and analyzed using Image J software [31 (link)].

Western bolt analysis was applied to detect the protein expression levels of apoptosis indexes and exosomal markers. The BCA protein assay was adopted as a standard to determine the protein content. Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked with 50% fat-free milk and incubated overnight with the appropriate primary antibodies including anti-Bcl-2 (sc-7382, 1:1000), anti-Bax (sc-7480, 1:1000), anti-β-actin (sc-8432, 1:5000) (SantaCruz Biotechnology, CA, USA), anti-cleaved caspase-3 (9668, 1:1000) and anti-cleaved caspase-9 (9509, 1:1000) (Cell Signaling Technology, Beverly, MA, USA). After fully rinsing with Tris Buffered Saline Tween containing 0.1% Triton×100 buffer solution, the membrane was incubated with secondary antibody (sc-69,786, 1:1000; Santa Cruz Inc, Santa Cruz) at room temperature for 1 h. Then, the protein bands were visualized by an Ultra High Sensitivity ECL Substrate Kit (ab133409, Abcam, Shanghai, China). The semi-quantitative results were obtained by quantification of optical density using the ImageJ software.

Dexamethasone sodium phosphate injection (specification: 1 ml: 5 mg, license number: NMPN H37021969) was purchased from Chenxin Pharmaceutical Incorporated Company. Gentamicin sulphate injection (specifications: 2 ml: 8 million units, license number: NMPN41025466) was purchased from Henan Furen Huaiqingtang Pharmaceutical Company Limited. Anti-p-PI3K (cat. no. 4228), anti-PI3K (cat. no. 4292), anti-p-AKT (cat. no. 4060), anti-AKT (cat. no. 4691), anti-p-TAK1 (cat. no. 9339), anti-TAK1 (cat. no. 4505), anti-p-IKKα/β (cat. no. 2697), anti-IKKα (cat. no. 11930), anti-IKKβ (cat. no. 2678), anti-p-IκBα (cat. no. 2859), anti-IκBα (cat. no. 4814) were purchased from Cell Signaling Technology, Inc. Anti-IL-1β (sc-12742), anti-VEGF (sc-7269), anti-AQP1 (sc-25287), anti-AQP3 (sc-518001), anti-Bcl-2 (sc-7382), anti-Bax (sc-7480), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology. Anti-NF-κB (ab16502), anti-PHLPP2 (ab71973) and anti-NEMO (ab178872) was purchased from ABCAM. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (cat. no. AP308P) and unconjugated goat anti-rabbit IgG (cat. no. AP132) were purchased from Sigma-Aldrich (Merck KGaA). DAB concentrated kits (PAB180021) was purchased from Bioswamp.