Mouse monoclonal antibody against human β actin

Cells were collected, washed with PBS, and then lysed with lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0, and 5 mM EDTA) containing protease inhibitors (10 μg/mL phenylmethylsulfonyl fluoride). After quantification with BCA assay (Beyotime Biotechnology, Beijing, China), 30 μg total proteins were loaded and separated on 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk, probed with rabbit polyclonal antibodies against human LC3 or cleaved LC3 (LC3-II) or Beclin 1 (Cell Signaling Technology, USA), mouse monoclonal antibody against human β-actin (Cell Signaling Technology, USA), and ASPP2 (sigma, Germany). The membranes were washed, probed with HRP conjugated secondary antibody, and detected with chemiluminescent kit. The western blot images were quantitatively analyzed with Image J software (National Institutes of Health, USA).

Protein concentrations from endothelial cells extracts were determined by the BCA method (Pierce BCA Protein Assay, Thermo Fisher Scientific, Waltham, MA, USA). Samples were denatured, subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. Non-specific binding sites were blocked with 5% BSA in TBS solution and membranes were incubated overnight with following primary rabbit polyclonal antibodies: CAR (dilution 1:500, cat# ab186869, Abcam Waltham, MA, USA); phosphorylated-p38 MAPK (dilution 1:500, Thr180/Tyr182, cat#9211, Cell Signalling, Beverly, MA, USA), p-38 MAPK (dilution 1:500, cat#9212, Cell Signaling, Beverly, MA, USA), phosphorylated-p65 (dilution 1:200, Ser536, cat# 3033, Cell Signalling, Beverly, MA, USA), NF-κB p65 (dilution 1:200, cat#4764, Cell Signalling, Beverly, MA, USA) and mouse monoclonal antibody against human β-actin (dilution 1:2000, cat# 3700, Cell Signalling, Beverly, MA, USA). Next, membranes were washed and incubated for 1 h with a secondary HRP-linked anti-rabbit antibody (dilution 1:2000, cat#0448, Dako, Glostrup, Denmark). The blots were developed using a chemiluminescence detection system. Signals were recorded using a luminescent analyzer (ImageQuant™ LAS 500, GE Healthcare, Uppsala, Sweden) and quantified with ImageJ software (NIH, https://imagej.nih.gov/ij/ (accessed on 22 July 2021)).