Ab53226

Cells were washed in phosphate buffered saline (PBS) twice before proteins were extracted, and proteins were separated on a SDS/PAGE gel, transferred onto a PVDF membrane and subjected to immunoblot analysis. Blotting was performed with antibodies against E-cadherin (ab53226, Abcam, Cambridge, UK), vementin (ab8978, Abcam, Cambridge, UK), ZEB1 (ab180905, Abcam, Cambridge, UK), ZNF217 (ab136678, Abcam, Cambridge, UK). Goat anti-rabbit and goat anti-mouse immunoglobulin horseradish peroxidase-linked F(ab)2 fragments (ZAGB-bio, Beijing, China) were used as secondary antibodies.

Primary AEC isolated from lung biopsy were fixed in 3% paraformaldehyde (PFA) for 10 minutes at room temperature, washed three times in 0.1% glycin/PBS, and incubated with either rabbit polyclonal antibody to E-cadherin (1:200) (#ab53226) (Abcam, USA), or rabbit polyclonal SP-C (1:50) (#sc13979) (Santa Cruz Biotechnology, USA) or mouse monoclonal antibody to surfactant protein A (SP-A) (1:200) (#ab51891) (Abcam, USA) over night in a moist chamber at 4 °C. For detection Cy3 labelled anti rabbit secondary antibody was used for E-cadherin and SP-C, while a alkaline phosphatase labeling was used to detect SP-A. A549 cells were treated with TN or TG, for 18 hours and 7 days respectively and grown in either BMSC-cm or normal media for 24 hours. Cells were then fixed in 3% PFA as described above and subsequently stained overnight at 4 °C in a moist box with anti-cytokeratin, pan-FITC anti-mouse (1:200) (#C5992), mouse anti-vimentin-Cy3 (1:1000) (both: Sigma, USA) (#C9080) or caspase-3 (1:200) (#9662 S) (Cell Signaling, USA). Samples were then washed 3 times with PBS and analyzed under a confocal microscope LSM 510 Carl Zeiss or under Leica DMI4000D microscope.