Protease inhibitor cocktail 100

KR-12 (KRIVQRIKDFLR) was synthesized and purified by GL Biochemistry (Shanghai). Alizarin Red dye solution was purchased from Servicebio (Wuhan, China). Phosphatase (ALP) Color Development Kit, ALP Quantitative Kit, RIPA lysis buffer, and phenylmethylsulfonyl fluoride (PMSF; 100×) were purchased from Beyotime Biotechnology (Nantong, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). The SYBR Premix EX Taq Real-time PCR kit and cDNA Synthesis Kit were purchased from TaKaRa (Japan). Protease inhibitor cocktail (100×) and the BCA Protein Assay Kit were purchased from Thermo Fisher (USA). Polyvinylidene fluoride membrane for western blotting and Immobilon western Chemiluminescent HRP Substrate (ECL) were purchased from Millipore (USA). Monoclonal antibodies against SMAD1 (D59D7), SMAD5 (D4G2), SMAD4 (D3M6U), P-SMAD1/5 (41D10), GAPDH (D16H11), and horseradish peroxidase-linked anti-IgG secondary antibodies were purchased from Cell Signaling Technology (USA). TGF-β/SMAD inhibitor (LDN-193189 HCL) was purchased from Selleck Chemicals (Houston, TX, USA). Lipofectamine™ 3000 and Lipofectamine™ RNAiMAX transfection reagent were purchased from Invitrogen (Carlsbad, CA, USA). Gaussia luciferase reporter plasmids and Secrete-Pair Assay Kit were purchased from Genecopoeia (Rockville, MD, USA).

The transfected control Caco2 cells were lysed in RIPA buffer (150 mM NaCl; 50 mM Tris-Cl, pH 8; 1% NP-40; 0.5% deoxycholate; 0.1% SDS) (Beyotime, Shanghai, China) with Protease Inhibitor Cocktail (100×) (Thermo Scientific, MA, USA). The resulting samples were separated using 10% SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 1 h in 5% non-fat milk in TBST at 25°C, the membranes were incubated overnight at 4 °C with specific primary antibodies, including SMARCE1 (Abcam, Cambridge, UK, 1:1000), tfec (Santa Cruz, USA,1:500), and β-tubulin (Sigma, USA, 1:10,000). Primary antibody binding was visualized using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Sigma, USA, 1:10,000) for 1 h at 25°C. Signal intensities were measured using a Chemiluminescent HRP substrate (Yamei, Shanghai, China) and imaged using ImageJ v1.8.0 (NIH, Bethesda, MD, USA).