Mouse kc elisa kit

CC16 antibody for human and monkey staining were purchased from BioVendor (Asheville, NC). CC16 antibody for mouse staining was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse KC ELISA kit was from R&D Systems (Minneapolis, MN).

Mouse lung epithelial (MLE)-12 cells were cultured in DMEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (10 μg/mL) at 37°C and 5% CO₂ in humidified air as described previously (34 (link)). These cells were exposed to hyperoxia (95% O₂ and 5% CO₂) for 72 hours. The cells were pretreated with vehicle, 4-PBA (0.3 mM), or claudin-4 siRNA (25 nM). The dose of 25 nM claudin-4 siRNA used in MLE-12 cell was determined by Western blot (Supplementary Figure 2). For claudin-4 siRNA transfection, MLE-12 cells were incubated for 24 hours and transfected with claudin-4 siRNA, using DharmaFECT™ 1 siRNA Transfection Reagent (Dharmacon Inc. Chicago, IL, USA). MLE-12 cells with non-targeting control siRNA treatment were used as negative controls. After 24 hours, the culture medium was replaced with the recommended medium. Forty-eight hours after claudin-4 siRNA treatment, MLE-12 cells were exposed to hyperoxia for 72 hours. Next, cells were lysed for protein extraction subjected to the western blot procedure. The supernatant was collected and assayed for KC using a mouse KC ELISA kit (R&D, Inc.). The experiments were performed in triplicates.