Mouse anti glyceraldehyde 3 phosphate dehydrogenase

Western blot assay was performed to determine the protein expression levels in each group. The cells were lysed in cold radioimmunoprecipitation assay buffer (Invitrogen Life Technologies, Carlsbad, CA, USA). A BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentrations, according to the manufacturer’s instruction. Subsequently, the proteins were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% fat dry milk in PBS for 4 h. Subsequently, the PVDF membrane was incubated with specific primary antibodies (mouse anti-cyclin D1, mouse anti-PCNA, mouse anti-CDK4, mouse anti-smooth muscle-α-actin, mouse anti-smoothelin, mouse anti-desmin, mouse anti-phospho-Akt, mouse-anti-Akt, anti-phospho-ERK, mouse-anti-ERK, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibodies; all from Abcam, Cambridge, UK) for 3 h. After washing three times with PBS (5 min each time), the PVDF membrane was incubated with a rabbit anti-mouse secondary antibody (Abcam). Next, after washing three times with PBS (5 min each time), an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific) was used to detect the immune complexes present on the PVDF membrane.