Epr16769

50 μg/well of protein from the HepG2 cell lysate was separate using SDS-PAGE and transferred to a nitrocellulose membrane (G.E. Bioscience). After blocking with 5% skim milk in PBST for 1 h at room temperature, membranes were incubated overnight with primary antibody at 4 °C. Layers were then washed with PBST and incubated with horseradish peroxidase (HRP)–conjugated secondary antibody before visualization with ECL plus (G.E. Bioscience). All antibodies, including NF-κB (Cell signaling D14E12, #Cat.8242), IκBα (Cell signaling L35A5, #Cat. 4814), poly-Ub (Enzo FK2, #Cat. BML-PW0150–0100), P21 (Cell Signaling 12D1, #Cat 2947), Actin (Abcam EPR16769, #Cat. Ab179467), lamin A (Abcam EPR4068, #Cat. ab108922), and tubulin (Abcam EPR13796, #Cat. ab210797) were diluted in 5% BSA buffer. The dilution ratio of each antibody followed the manufacturer’s recommendation.

RT-PCR, RT-qPCR, and western blot analysis were performed as described previously.^{40 (link)} Total RNA was extracted from cultured cells using TRIzol RNA Isolation reagent (Takara, Tokyo, Japan), according to the manufacturer’s instruction. Three independent cultures were used for RNA preparations. First-strand cDNA was generated with High-Capacity cDNA Reverse Transcription Kit, (Applied Biosystems, San Diego, CA), and one microliter of each RT reaction mixture was amplified with Ex Taq DNA polymerase (Takara, Tokyo, Japan). As for RT-qPCR, cDNA was amplified using premix SYBR Green Ex Taq reagent kit (DRR820A, Takara) with a STEP ONE PLUS real-time PCR system (Applied Biosystems, Forster City, CA), according to the manufacturer’s instruction. All the primers used in this study are listed in Table 1. As for western blotting, anti-Dlx2 (1:800; ab85995, Abcam, Cambridge, UK), anti-OCN (1:1 000; ab93876, Abcam), and anti-β-actin (1:3 000; EPR16769, Abcam, Cambridge, UK) were used for the detection of Dlx2, OCN, and β-actin, respectively. The secondary antibodies used this study were bought from Sigma-Aldrich and conjugated to horseradish peroxidase (anti-rabbit (1:5 000, A0545) or anti-mouse (1:5 000, SAB3701214)).

Proteins were extracted using RIPA Lysis Buffer (Thermo Fisher Scientific, MA, USA) containing protease inhibitors cocktail (Cell Signaling Technology, MA, USA). Protein concentration was measured using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Then, protein extract (20 μg) was separated by SDS-PAGE and transferred onto polyvinylidene fluoride microporous membranes (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% milk in 0.5% Tris-buffer saline solution (pH 7.6) for 1 h, then incubated overnight at 4 °C with primary antibodies for CXCR4 (abcam; UMB2, 1:1000), GAPDH (abcam; EPR16891, 1:5000), CD9 (abcam; EPR2949, 1:2000), CD63 (Santa Cruz; MX-49.129.5, 1:200), TSG101 (abcam; EPR7130(B), 1:1000), or actin (abcam; EPR16769, 1:5000). The membrane was then incubated at room temperature for 30 min with HRP-conjugated secondary antibodies. The image data were collected on Bio-Rad molecular Imager with Image Lab^TM Software and analyzed with NIH ImageJ.