Mouse anti smoothelin

Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Recombinant human PDGF-BB was purchased from ACROBiosystems, Inc. (Newark, DE, USA) and AS-IV was purchased from Tauto Biotech (Shanghai, China). Dimethyl sulfoxide (DMSO) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-smoothelin, anti-α-smooth muscle actin (α-SMA), anti-desmin, anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-p38 MAPK, anti-matrix metalloproteinase (MMP)2, anti-MMP9 and anti-GAPDH antibodies, as well as a goat anti-mouse secondary antibody, were obtained from Abcam (Cambridge, UK).

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.