β actin antibody

NPTr monolayer cells were treated with either P. multocida or PBS, as described above in section Cell culture and bacterial infection. Challenged cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with a protease inhibitor cocktail (Sigma-Aldrich, Burlington, MA, USA), sonicated, and then centrifuged at 10,000 × g for 10 min at 4°C. The insoluble debris was removed and the protein concentration in the supernatant measured using a BCA protein assay kit (Beyotime, Shanghai, China). The cell lysates were then separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked in 5% BSA in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature and then incubated overnight with either β-catenin polyclonal antibody, E-cadherin polyclonal antibody, or β-actin antibodies (all from Proteintech, Wuhan, China). After washing, the blots were incubated with species-specific horseradish peroxidase-conjugated antibodies and finally visualized with enhanced chemiluminescence (ECL) reagents (Beyotime, Shanghai, China). All Western blots were densitometrically quantified using ImageJ software, and the results were analyzed as the relative immunoreactivity of each protein normalized to the respective loading control.

ICA (purity > 98%) was purchased from Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, China). 6-OHDA was obtained from Sigma-Aldrich (St. Louis, MO). Sytox green nucleic acid fluorescence stain was bought from Bio-Rad (Hercules, CA, USA). Anti-tyrosine hydroxylase (TH), HO-1, NQO1, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-Kelch-like ECH-associated protein 1 (Keap1), Nrf2, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule-1 (Iba-1), glia fibrillary acidic protein (GFAP), inducible nitric oxide synthase (iNOS), TNF-α, and β-actin antibodies were bought from Proteintech Group (Chicago, IL, USA). Nrf2-siRNA and control-siRNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The expression levels of BAP1, SLC7A11, GPX4, ASCL4, TNF-α, IL-1β and IL-6 were detected using western blot analysis. Briefly, total protein was extracted using RIPA lysis buffer (Beyotime, China), and a BCA kit (NCM Biotech, China) was used to determine protein concentration. The loading volume was determined to be 20 µg as the loading amount. SDS-PAGE gel electrophoresis was performed to separate the proteins, which were then transferred to a PVDF membrane (Millipore, USA). Next, After blocking with 5% skim milk powder (BD, USA), the membrane was incubated with BAP1, SLC7A11, GPX4, ASCL4, TNF-α, IL-1β and IL-6 and β-actin antibodies (Proteintech, China) overnight at 4℃. The corresponding secondary antibody (Bioss) was incubated at room temperature for 1 h on the day after washing the membrane. Finally, a chemiluminescence imaging system (Tanon, China) was used for development, and the results were analyzed using the ImageJ software for grayscale analysis.

CYC27 was synthesized and identified by our lab according to the previously reported procedure [16 (link)] (purity 98%). Rosiglitazone and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM, horse serum and penicillin–streptomycin were obtained from Hyclone (South Logan, UT, USA). FBS was purchased from PAN (Adenbach, Bavaria, Germany). RIPA lysis buffer and PMSF were bought from Solaibo (Beijing, China). Antibodies directed against IRS1, InsRβ and p-Akt (Ser473) were bought from Cell Signaling Technology (Danvers, MA, USA). Phospho-IRβ (Tyr1185) antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies against phospho -IRS1 (Tyr608) and PVDF membrane were bought from Millipore (Bedford, MA, USA). β-actin antibodies and all secondary antibodies were purchased from the Proteintech Group (Wuhan, Hubei, China). The BCA Protein Assay Kit was bought from Beyotime Biotechnology (Shanghai, China). Clarity^TM Western ECL Substrate was purchased from Bio-Rad (Hercules, CA, USA). The GHb, GSA, TC and TG assay kit were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).