Ab214666

The interaction between USP10 and KLF4 was detected by Co-IP. BMSCs were lysed using Co-IP lysate to extract proteins. Protein supernatants were incubated with Normal rabbit IgG or USP10 (AB70895, Abcam), and SKP2 (AB214666, Abcam) antibodies at 4℃ overnight with rotation. The Co-IP lysate was then added to Protein A/G agarose beads, mixed, and centrifuged to collect the precipitate. Following antibody incubation, the cell lysate was fully incubated with agarose beads for 2 h at 4℃ to allow antibody coupling to the beads. The agarose beads were washed using Co-IP lysate buffer, and WB subsequently analyzed the coupling product.

Western blots were performed to verify the expression of proteins in control and IPF group. Total protein extracts (20 μg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with anti-KLF4 (Abcam, Ab214666), anti-TERT (Abcam, Ab32020) and anti-GAPDH (Abcam, Ab8245) antibodies. The signal was detected by enhanced automatic chemiluminescence camera (Tanon 5200).

The total proteins of the cells were isolated by a radioimmunoprecipitation assay buffer (P0013E, Beyotime, Shanghai, China) and then quantified by a bicinchoninic acid protein assay kit (P0012, Beyotime, Shanghai, China). After the loading buffer was added, the proteins were denatured in boiling water. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinyl fluoride membrane (Millipore, Bedford, MA, United States). After that, the membrane was blocked in 5% skim milk for 30 min at room temperature. Next, the proteins were incubated with primary antibodies anti-KLF4 (ab214666, Abcam, Cambridge, United Kingdom) and anti-GAPDH (ab8245, Abcam, Cambridge, United Kingdom) overnight at 4°C and then incubated with the secondary antibody (ab6789, Abcam, Cambridge, United Kingdom) at room temperature for 1 h. Lastly, the protein bands were visualized using ECL chemiluminescence solution (P0018FS, Beyotime, Shanghai, China), and then, the gray value of the bands was quantified by Image Lab^TM Software.

Cell pellets were lysed with RIPA buffer (AR0102, BOSTER) with protease inhibitor added on ice. The protein concentration was determined according to the instructions of the Pierce™ BCA Protein Assay Kit (23227, Thermo). Equal amounts of protein were loaded onto 10% SDS-PAGE gels, separated by electrophoresis and transferred onto PVDF membranes (IPVH00010, Millipore). After blocking with 5% skim milk in TBST for 1.5 hours at room temperature, the membranes were incubated with rabbit anti-human Col1 (ab138492, Abcam), rabbit anti-human Col3 (ab184993, Abcam), rabbit anti-human KLF4 (ab214666, Abcam), rabbit anti-human α-SMA (ab7871, Abcam), rabbit anti-human BMP4 (ab124715, Abcam), and rabbit anti-human GAPDH (60004-1-lg, Proteintech) antibodies overnight at 4 °C. The next day, the membranes were incubated with IgG-HRP secondary antibody (A0208, Beyotime) for 1 h at 37 °C. Traces of immunoreactivity on the membrane were detected by ECL reagent (WBULS0100, Millipore) on a Fluor Chem FC system (Alpha Innotech). Bands were quantitated by ImageJ software.