Ba0761

HUMSCs, lysed in RIPA buffer, and frozen lung tissues, ground in a mortar with liquid nitrogen, were all stored aliquots at −80°C until assayed, respectively. Lysates were mixed with an equal volume of sample buffer, denatured by boiling, and then separated on a 10–15% polyacrylamide minigel. The proteins were transferred to nitrocellulose membranes, blocked with 5% milk, and incubated overnight with rabbit polyclonal anti-CXCR4 antibody (for HUMSC sample, 1 : 100, Boster, China, BA0761), mouse monoclonal anti-CXCR4 antibody (for lung tissue samples, 1 : 500, Abcam, USA, ab58176), or mouse monoclonal anti-TGF-β1 antibody (1 : 1000, Abcam, USA, ab64715) at 4°C. And then the blots were incubated with goat-anti-rabbit IgG HRP antibody (1 : 1000, Applygen Technologies Inc., China, C1309) or goat-anti-mouse IgG-HRP antibody (1 : 5000, Abcam, USA, A4416) for 1 h at room temperature. The membranes were rinsed with washing buffer and incubated with chemiluminescence (ECL) working solution for 5 min. The signals were detected by Gel software, and the relative intensities of the detected bands compared between groups.

The CXCR4-overexpressing HUMSCs or control HUMSCs, grown on glass coverslips, and frozen mouse lung tissues, sectioned at 4 μm and placed onto glass slides, were incubated overnight with rabbit polyclonal anti-CXCR4 antibody (1 : 200, Boster, China, BA0761), or rabbit polyclonal anti-pro-SP-C antibody (1 : 250, Abcam, USA, ab40879), respectively, in darkness at 4°C, followed by incubations with Dylight 549 (1 : 100, Abbkine, USA, A23320) for 1 h at 37°C. Then the total cell nuclei of the HUMSCs and the lung tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The fluorescence images of EGFP, CXCR4, and pro-SP-C were detected using a confocal laser scanning microscope (Leica, Germany, TCS SP8).