Pvdf membrane

The recombinant protein was mixed with loading buffer, boiled at 100 °C for 5 min, centrifuged at 12,000× g for 1 min, and separated by 12% SDS-PAGE gel before being transferred to a PVDF membrane (Burlington, MA, USA) by a trans-blot SD semi-dry electrophoretic transfer cell (Beijing Liuyi, China). Then, the PVDF membrane was incubated with 5% BSA for 2 h, primary antibody (Anti-His Tag Mouse Monoclonal Antibody, Abcam, Cambridge, UK) overnight, and secondary antibody (HRP Goat Anti-Mouse lgG, Abcam, Cambridge, UK) for 2 h. Each time the incubation solution was changed, 1× TBST was used to wash the PVDF membrane. Finally, the target band on the PVDF membrane was dyed by using the Pierce™ ECL Plus Western blotting substrate (ThermoFisher, Waltham, MA, USA) and detected by using the chemiluminescent imaging system.

The expression levels of the NPPA, TPM1, and TPM2 in the control and model groups were measured by Western blotting with glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal control protein. An aliquot (40 µg) of protein extract from the heart tissue of each group was boiled at 95°C for 5 min in SDS-loading buffer and separated over 4–12% polyacrylamide gel (GeneScript) at 140 V for 1 h with Tris-MOPS-SDS running buffer (GeneScript). The proteins were then transferred to the PVDF membrane by semi-dry transfer blotting at 20 V for 1 h. The PVDF membrane (Millipore) with proteins was cut into two parts along ca. 30 kD, and then blocked in 5% milk TBST buffer for 120 min. Anti-NPPA antibody (Abcam), anti-TPM2 (Abcam), anti-TPM1 (Abcam), and anti-GAPDH (Abcam) were diluted at 1:1000, 1:300, 1:1000, and 1:5000, respectively, to blot PVDF membrane by incubation at 4°C for 10 h, and then washed three times by 5% milk TBST for 5 min each. The PVDF membrane was then incubated with horseradish peroxidase-conjugated second antibodies, goat antirabbit antibody (Abcam) for NPPA, TPM2, and TPM1 and goat antimouse antibody (Abcam) for GAPDH, respectively, for 1 h at 25°C. After washing with TBST, the protein bands were visualized by enhanced chemiluminescence (Tanon 5200Multi, China), and the optical densities were quantified using ImageJ.

The cells were grown into six-well cell culture plates at 4 × 10⁵ cells/well and after 36 h of transfection, they were lysed with RIPA lysis and extraction buffer as described by the manufacturer (Thermo Fisher Scientific Inc, Rockford, IL, USA). The proteins were separated on 8% SDS-PAGE gels and electrotransferred to PVDF membrane (Millipore Co., Billerica, MA, USA). The PVDF membrane was blocked with 5% non-fat milk in TBS-T (0.15 mM sodium chloride, 0.01 mM Tris-base and 0.1% tween-20, pH 8.0) for 3 h, the PVDF membrane was incubated with rabbit anti-GFP polyclonal antibody (Abcam, Cambridge, UK) diluted in TBS-T(1:1000)overnight at 4 ℃. After washing three times with TBS-T, the PVDF membrane was incubated with DyLight 800 goat anti-rabbit IgG (Abbkine, Wuhan, China) at 1:8000 dilution in TBS-T. Finally, the membrane was washed three times with TBS-T, and bands were scanned using the Odyssey system (LI-COR Bioscience, Lincoln, NE, USA).

Western Blotting was carried out as previous described [24 (link)]. Briefly, cells were lysed in cold RIPA buffer (Life Technologies). The protein concentration was determined using the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). Protein was separated with 10% SDS-PAGE, transferred to a PVDF membrane (Life Technologies), and then blocked in 5% nonfat dried milk (Yili, Beijing, China) in DPBS (Life Technologies) for 4 h. The PVDF membrane was then incubated with rabbit anti-YAP1 monoclonal antibody (1:100, Abcam, Cambridge, MA, USA), or mouse anti-GAPDH monoclonal antibody (1:100, Abcam) as an internal reference for 3 h at room temperature, and then washed by DPBS for 10 min. After that, the PVDF membrane was incubated with mouse anti-rabbit secondary antibody (1:20000, Abcam) for 1 h at room temperature. After washing by DPBS for 15 min, the immune complexes on PVDF membrane was detected using the ECL Western Blotting Kit (Pierce Chemical, Rockford, IL, USA). Image-Pro plus software 6.0 was used to analyze the relative protein expression, represented as the density ratio versus GAPDH.