Mouse anti cyclin d1

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.

PRF was obtained from Anhui Joyfar Pharmaceutical Co., Ltd, (Bozhou, China), while Dulbecco’s modified Eagle’s medium (DMEM)/F12 and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), MTT and recombinant human PDGF-BB were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-cyclin D1, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase (CDK) 4, α-SMA, desmin, smoothelin, phospho-protein kinase B (Akt), total Akt, phospho-ERK, total ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and rabbit anti-mouse secondary antibodies were obtained from Abcam (Cambridge, UK).

The human colorectal cancer cell lines, HT29 and HCT116, were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and stored in the Key Laboratory of Cell Differentiation and Apoptosis of the National Ministry of Education (Shanghai, China). NDRG1 over-expressing and silenced clones of the colorectal cancer cell lines, HT29 and HCT116, were constructed, as previously described [18 (link)]. Both HT29 and HCT116 cells were cultured in McCoy's 5A medium (Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA). Cells were cultured in humidified air with 5% CO₂ at 37°C.
The primary antibodies used were as follows: goat anti-NDRG1, mouse anti-cyclin D1, rabbit anti-lamin B1, mouse anti-Na⁺-K⁺ATPase β2 (Abcam, USA); rabbit anti-β-catenin, mouse anti-CD44 (Cell Signaling Technology, Inc., USA); mouse anti-c-Myc, mouse anti-β-actin (Santa Cruz Biotechnology, USA); Phycoerythrin (PE)-labeled anti-human CD44 (BD Pharmingen™, USA) and PE-labeled anti-human CD133/1 (AC133) (Miltenyi Biotec, Germany).