Anti pcna

Cells were treated with a series concentrations of Huaier (0, 5, 10 and 15mg/ml) for 24h, and then lysed by cell lysis buffer for Western and IP (Beyotime, P0013). Protein concentration was detected with BCA Protein Assay Kit (KeyGen BioTECH, KGP902). After that, 20µg proteins were separated by 10% or 12% SDS-PAGE and transferred onto Pure Nitrocellulose Blotting Membrane (Pall Corporation, P/N 66485). Then, the blots were blocked for nonspecific binding with 5% non-fat milk in PBST (PBS, Tween-20, pH7.4) at room temperature for 1h. Next, the blots were incubated overnight at 4℃ with 5% non-fat milk containing primary antibodies which are listed as follows: anti-PCNA (ImmunoWay, YM3031), anti-Ki-67 (ImmunoWay, YT2467), anti-β-actin (ImmunoWay, YM3028), anti-Bcl-2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-E-cadherin (ImmunoWay, YT1454), anti-N-cadherin (ImmunoWay, YT2988), anti-YAP1 (abcam, ab52771), anti-p-YAP1 (ab76252), anti-CyclinD1 (abcam, ab134175), anti-Cleaved Caspase Substrate Motif (Cell Signaling, #8698) and anti-YAP/TAZ (Cell Signaling, #8418). After that, incubating blots with secondary antibodies conjugated with HRP (Cell Signaling, #7074 or #7076) for 1h at room temperature. Finally, after washing three times with PBST, the blots were visualized by New Super ECL Assay (KeyGen BioTECH, KGP1128).

VSMCs were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Honokiol was purchased from Selleck Chemicals (Houston, USA, catalog no. S2310), MSNPs (catalog no. 643645) and F‑127 pluronic gel were purchased from Sigma‑Aldrich Co. (St Louis, MO, USA). Transmission electron microscope was acquired from FEI (Hillsboro, Oregon, USA). FBS and 0.25% (w/v) trypsin−EDTA were obtained from Corning (New York, USA). DMEM and penicillin−streptomycin solution were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Culture flasks (25 cm² surface area), culture plates, a filter of 8 μm pore size and confocal dishes were acquired from Corning Incorporated (Corning, NY, USA). 2F Fogarty embolectomy catheters were obtained from Edwards Lifesciences (Irvine, CA, USA). WST-1 was purchased from Beyotime Biotechnology (Shanghai, China). Foxp3/Transcription Factor Staining Buffer Set was purchased from Thermo Fisher Scientific. The primary antibodies used in Western blotting analysis were: anti-p-smad3, anti-p-ERK1/2 and anti-PCNA (Immunoway Biotechnology, NY, USA). α‑SMA antibody was obtained from Abcam (Cambridge, UK).