Super ecl plus detection kit

To detect the effects of MPNCR and miR-31 on their target genes at the protein level, a Western blot assay was performed. The total protein was extracted from BMECs transfected with the overexpression vector pcDNA3.1(+)-MPNCR (or pcDNA3.1(+)) (600 ng/mL), miR-31 mimic (or mimic-NC) or miR-31 inhibitor (or inhibitor-NC) (20 nM) for 72 h, using radioimmunoprecipitation assay (RIPA) cell lysis solution (Applygen, Beijing, China). The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred onto a poly (vinylidene di uoride) (PVDF) membrane (Millipore Corporation, MA, USA). Then, the membranes were sealed with 5% skimmed milk solution for 1 h at room temperature and incubated at 4 °C for 12 h with the primary antibody against CAMK2D (Abcam, Cambridge, UK). Then the PVDF membrane was incubated with an HRP conjugated secondary antibody goat-anti-rabbit IgG (Zhongshan-Bio, Beijing, China) for 2 h. After washing three times with PBS, the protein bands were observed with a Super ECL Plus detection kit (Applygen, Beijing, China) using a Mini Chemiluminescent Imaging and Analysis System (Beijing Sage Creation Science Co., Beijing, China). Densitometry analysis was performed using Sage Capture software (Beijing Sage Creation Science Co.) using GAPDH (Abcam) as a control.