Trans blot unit

For Western blot analyses, cell extractions were cleared by 13300 × g for 15 min at 4°C. Protein concentration was determined by BCA Protein Assay Kit (APPLYGEN, Beijing, China). The protein samples were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane using a Trans-blot unit (Bio-Rad Laboratories) for 1.5 hour at 250 mA. The membranes were blocked with 5% (wt/vol) skim milk and 0.1% (vol/vol) Tween-20 in TBS (pH 7.4) for 1 hour at room temperature (25°C). Primary antibodies against BMP4 (1 : 500, ab39973, Abcam, UK), p38 MAPK (1 : 200, sc-7149, Santa Cruz Biotechnology, USA), phospho-p38 (p-p38) MAPK (1 : 1,000, 9216, Cell Signaling Technology, USA), Sgk1 (1 : 500, ab59337, Abcam, UK), p-Sgk1 (1 : 1,000, 44-1260G, ThermoFisher, USA), Nedd4-2 (1 : 500, 4013, Cell Signaling Technology, USA), p-Nedd4-2 (1 : 500, ab168349, Abcam, UK), and GAPDH (1 : 5,000, ab8245, Abcam, UK) were incubated with the membranes overnight at 4°C. After washing with TBS-T, the membranes were incubated for 1 hour at room temperature with the corresponding secondary antibodies (1 : 10,000). All membranes were washed with TBS-T, and the bands were quantified by using the Odyssey infrared imaging system (LI-COR) and Odyssey v3.0 software.

The samples were run on 12% SDS-polyacrylamide gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After electroblotting at 35 volts for 16 hours in transfer buffer using a Transblot unit (Bio-Rad), the target protein was blocked by incubating for 1 hour in TBST containing 3% (w/v) BSA (Sigma-Aldrich), followed by detection with specific mouse anti-IL-18 (R&D systems) in 1:3,000 dilution and incubated at room temperature for 1 hour. The antibody was removed and the PVDF membrane was washed three times for 5 min each in TBST with gentle agitation. Horse radish peroxidase-conjugated goat anti-mouse (R&D system) was added at a dilution of 1:10,000 in TBST containing 3% (w/v) BSA and incubated for 1 hour with gentle agitation at room temperature. The sheet was then washed three times in TBST and antigen-antibody complexes were detected by the addition of LuminataTM Forte Western HRP substrate (Millipore).