Bca protein detection kit

The total protein was obtained from the thoracic aorta and left ventricular of three rats selected randomly using RIPA lysis buffer with PMSF and phosphatase inhibitor (Servicebio, China). Protein concentration was measured using a BCA protein detection kit (Servicebio, China). SDS-PAGE separated the whole protein, which was subsequently transferred to 0.45 μM on the PVDF membrane (Servicebio, China). The membrane was sealed with 5% nonfat milk for 1 hour at room temperature before being incubated with the primary antibody overnight at 4°C. The membrane was then washed three times with TBST, incubated for one hour at room temperature with a secondary antibody, and the protein was identified using an ECL reagent (Servicebio, China). Primary antibodies include TAK1 (GB11564, 1: 1,000, Servicebio, China), IKB-alpha (GB13212-1, 1 : 1,000, Servicebio, China), p65 (GB11997, 1 : 1,000, Servicebio, China), and β-actin (GB15001, 1: 2,000, Servicebio, China).

Total protein was obtained from the left ventricular of three rats selected randomly using RIPA lysis buffer with PMSF and phosphatase inhibitor (Servicebio, China), and protein concentration was measured using a BCA protein detection kit (Servicebio, China). SDS-PAGE was used to separate the whole protein, which was subsequently transferred to 0.45 μM on the PVDF membrane (Servicebio, China). Before incubating with the primary antibody overnight at 4°C, the membrane was sealed for one hour at room temperature with 5% nonfat milk. The membrane was then washed three times with TBST, incubated with a secondary antibody for one hour at room temperature, and the protein was identified using an ECL reagent (servicebio, China). Primary antibodies include: TGF-beta 1 (bsm-33287m, 1 : 1,000, Bioss, China), Ras 1 (GB11411, 1 : 1,000, Servicebio, China), ERK1/2 (GB11560, 1 : 1,000, Servicebio, China), C-Fos (GB114125, 1 : 1,000, Servicebio, China), and β-actin (GB15001, 1 : 2,000, Servicebio, China).

GP was homogenized in ice-cold PBS, stored at 4 °C for 2 h. Later, the supernatant was collected after the centrifugation at 14,000 x g for 10 min. Total protein concentration was determined by BCA protein detection kit (Service Biotechnology, Wuhan, China) and the samples were stored at − 70 °C. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) until the dye band reached the end of gel (10% polyacrylamide gel) and was transferred on polyvinylidene difluoride (PVDF) membrane. These membranes were incubated in 5% skimmed milk for 1.5 h at room temperature. Subsequently, the membranes were incubated at 4 °C for overnight with rabbit monoclonal anti-BMP-2 primary antibodies (1:1000 dilution). Membranes were washed with tris-buffred saline tween (TBST) with a time span of 5 min for each time. Membranes were incubated at room temperature for 30 min with secondary antibody (1:3000 dilution) and were washed again with TBST 4 times. Finally, after washing the images were taken using an imaging system (UVP, Upland, CA, USA).