Ecl system

Cultured cells were lysed in RIPA buffer (Beyotime Inc., NanTong, China). Protein concentration was identified using the Bradford reagent (Beyotime Inc.). We then performed electrophoresis of protein extracts and subsequent blotting as previously described [91 (link), 92 (link)]. In brief, Equivalent amounts of protein (30 μg) were separated by SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were then immunoblotted with the appropriate primary antibodies against Smad3 or p-Smad3 (Santa Cruz Biotechnology, Santa Cruz, CA), at 4°C for overnight, and subsequently were incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibodies at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) system (Beyotime, China) on Kodak X-ray film. Equal protein loading was assessed by the expression of β-actin. The protein bands were quantified using the BioRad Quantity One software package.

Western blot analyses were performed to evaluate target protein expression. Briefly, protein lysates (~20 μg per sample) were loaded and run on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to NC membranes (Millipore Corporation, MA, USA), and blocked with 5.0% nonfat milk for 3 h. The membranes were incubated with primary antibodies overnight at 4°C and then washed with Tris-buffered saline Tween-20 (TBST) three times for 10 min. Afterwards, membranes were incubated with the secondary antibodies for 2 h at room temperature and washed again. The bands on the membranes were visualized using an ECL system (Beyotime Institute of Biotechnology). Finally, membranes were exposed with an Odyssey infrared imaging system (LI-COR Biosciences, NE, USA). The following antibodies were used: anti-human FOLR1 (1:5000 dilution; Abcam, Cambridge, UK; CAT# ab221543), anti-human PCFT (1:300 dilution; Santa Cruz Biotechnology, Dallas, TX; CAT# sc-393460), anti-human RFC (1:300 dilution; Santa Cruz Biotechnology; CAT# sc-390948), anti-human GAPDH (1:5000 dilution; Abcam; CAT# ab181602), and HRP-labeled secondary antibodies (1:5000 dilution; Proteintech Group, Rosemont, IL).

The cells and tissue samples were lysed in RIPA buffer (Solarbio, #R0020, China) supplemented with protease inhibitors. Equal amounts of protein were separated with 10% SDS polyacrylamide gel (YEASEN, #20325ES62, China) and then transferred to PVDF membranes (Millipore, #IPVH00010, USA). After blocked with 5% skim milk at room temperature for 1 hour, the membranes were subject to relevant primary antibodies specific for Olig2 (1:1000, Affinity #DF8004, USA), CD133 (1:2000, Affinity, #AF5120, USA), Nanog (1:2000, CST, #3580 S, USA), Oct4 (1:2000, CST, #2750 S, USA), Flag (1:2000, Affinity #T0003, USA), Ubiquitin (1:2000, CST, #3936, USA), β-actin (1:5000, Sigma, #AF441, USA) at 4 °C overnight and then incubated with respective second antibodies. The membranes were developed with the ECL System (Beyotime, #P0018S, China).

HGFs were cultured onto each sample in 24-well plates at a density of 5 × 10⁴ cells per well with or without CM for Western blot experiments. After 7 days of culture, HGFs were lysed in RIPA buffer and proteins were separated on 8% SDS-PAGE gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes which were blocked for 1 h by QuickBlock buffer (Beyotime, Jiangsu, China) at room temperature. Membranes were washed in TBST and probed overnight at 4°C with one of the following primary antibodies: collagen-I (ab34710, 1:2,000; Abcam, USA), vinculin (ab129002, 1:10,000; Abcam, USA), fibronectin (ab2413, 1:1,000; Abcam, USA), and β-actin (1:1,000, ab8227; Abcam, USA). Membranes were washed in TBST and incubated for 1 h with HRP-conjugated secondary antibodies at room temperature. Proteins were visualized using the ECL system (Beyotime, Jiangsu, China).