Sftpc

Proteins were extracted and separated on a 10% SDS-PAGE gel ahead of being transferred on PVDF membranes (Life Technologies, USA). Then, membranes were blocked in 5% BSA-TBST for 2 h and probed with different primary antibodies overnight such as Angiopoietin 1 (Abcam, ab102015, UK), GLP-1R (Novus, NBP1-97308SS, USA), p-β-catenin (Santa cruz, sc-57535, USA), TCF7L2 (Abways, CY5720, China), SFTPC (Abclonal, A11764, USA), β-catenin (Abways, CY3523, China), FGF10 (Abclonal, A1201, USA), PKA C-α (Cell signaling, D38C6, USA). GAPDH (Santa cruz, sc-166574, USA) or PCNA (Abways, AB0051, China) was used as a loading control of total and nuclei protein. An anti-rabbit HRP secondary antibody was used for detection with the ECL technique.

ChIP assay was performed using EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, upstate) according to the manufacturer’s instructions. Briefly, cells cultured under the indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 ml of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from soluble chromatin preparations were approximately 400–800 bp in length. Immunoprecipitation (IP) was carried out overnight with Angiopoietin 1 (Abcam, ab102015, USA), SFTPC (Abclonal, A11764, USA), FGF10 (Abclonal, A1201, USA), or normal mouse IgG as a negative control. Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed with 2 μl DNA samples with the following primers: SPC: forward, 5′-AAGAGATCCCTCTCCCAGCA-3′; reverse, 5′-TGGGGTTTGCCGCCATC-3′; Ang-1: forward, 5′-AACAATTTCTCCTTTGATAGGTGGT-3′; reverse, 5′-GCCTTTCCGGATATCATGACC-3′;
FGF-10: forward, 5′-TCGCCATAAAGTGCGTTTGC-3′; reverse, 5′-GCCCTTCACTGAATCATGCG-3′.