β actin ab8226

Mouse lung tissue and MLE-12 cells were lysed by RIPA lysate. Mouse lung tissue needs to be additionally put into a homogenizer to fully lyse. The protein lysate was then centrifuged (12,000 g, 15 min, 4°C) and the supernatant was collected. After being mixed into the loading buffer, the protein lysate was heated to 100°C for 5 min. Then, we made a 10% sodium dodecyl sulfate polyacrylamide gel and added 10 μL of protein lysate. After electrophoresis and membrane transfer, the protein is transferred to the polyvinylidene fluoride membrane. We used 5% bovine serum albumin-PBS-Tween to block nonspecific antigens for 1 h at room temperature. The membrane was then incubated with primary antibody dilutions (β-actin, ab8226, Abcam, Cambridge, MA, USA; IL-1β, ab254360, Abcam, Cambridge, MA, USA; tumor necrosis factor (TNF), ab1793, Abcam, Cambridge, MA, USA; SOD1, ab51254, Abcam, Cambridge, MA, USA; SOD2, ab68155, Abcam, Cambridge, MA, USA; caspase 3, ab184787, Abcam, Cambridge, MA, USA; p65, ab16502, Abcam, Cambridge, MA, USA; p-p65, ab86299, Abcam, Cambridge, MA, USA; IκBα, ab183503, Abcam, Cambridge, MA, USA;) at 4°C overnight. After washing the membrane, we incubated the membrane at room temperature for 1 h with the secondary antibody dilution (Abcam, Cambridge, MA, USA). Finally, we used chemiluminescent liquid for color development.

Frozen PCa tissue was obtained with informed consent from patients who underwent radical resections at the Shandong Provincial Hospital. Ethical consent was granted by the Committees for Ethical Review of Research involving Human Subjects of Shandong Provincial Hospital. LNCaP, C4-2, DU145, and PC3 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum and kept at 37°C and 5% CO₂. The normal prostate cell line RWPE-1 was cultured in keratinocyte serum free medium (GIBICO, 17005–042, Grand Island, NY. Monoclonal TGF-β1 (ab9758), E-cadherin(ab76055),N-cadherin (ab18203), Cyclin D1 (ab137875), and β actin (ab8226) antibodies were got from Abcam (Cambridge, MA, USA). LY2109761 (No: S2704) was purchased from Selleck (Houston, TX, USA).

FAC (E0375), Arecoline (S2614), Fer-1 (S7243), NAC (S1623), and GSH (S4606) were purchased from Selleck (Shanghai, China). Calcein was purchased from Dojin (Tokyo, Japan). The Cell Counting Kit-8 (CCK-8) assay kit was also purchased from Dojin (Japan). Maleic dialdehyde (MDA), DCFH-DA assay kits, Alizarin red S, alkaline phosphatase (ALP), and diethanolamine activity kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against HO-1 (ab189491), GPX4 (ab125066), SLC7A11 (ab175186), and β-actin (ab8226) were purchased from Abcam (Cambridge, MA, USA). MC3T3-E1 cells (Procell Life Sci.&Tech. Co., Ltd., Wuhan, China) were incubated in MEM with 10% FBS, 10 mM glutamine, and 100 U/mL penicillin/streptomycin. For osteogenic differentiation, the culture medium was replaced with MEM containing 10% FBS, 10 mM β-glycerol phosphate, 50 μM ascorbic acid, 100 nM dexamethasone, and 100 U/mL penicillin/streptomycin.