Protease mixture inhibitors

Protein was isolated from murine lung tissue and HPMECs using RIPA buffer (Thermo Fisher) containing protease mixture inhibitors (Thermo Fisher). Proteins were separated by 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to a PVDF membrane using a Mini-PROTEAN Tetra Cell system (Bio-Rad). The following primary antibodies were used: goat anti-Snai1 (1 : 1000, Abcam, ab53519), rabbit anti-β-actin (1 : 5000, Cell Signaling, 4970), and rabbit anti-vinculin (1 : 1000, Cat#4650, Cell Signaling). Pierce ECL plus a Western blotting substrate (Cat #32132, Thermo Fisher) was used for visualizing immunoreactive protein bands. The protein bands were normalized by β-actin or vinculin on the same membrane.

Collected cells were resuspended in cold lysis buffer (50 mM TrisHCl at pH 7.5, 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 50 mM β-glycerol-phosphate, 0.1% NP 40, and 10% glycerol) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitors mixture (Thermo Scientific, Waltham, MA, USA). Cells were lysed using glass beads, and the protein concentration of the extracts was measured at 280 nm and normalized with lysis buffer. For immunoprecipitation, 200 µL of extracts were incubated with 40 µL of FLAG-Sepharose beads (Sigma-Aldrich) overnight at 4 °C. Beads were extensively washed with alkaline phosphatase buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 10 mM MgCl₂, and 1 mM DTT, adjusted to a pH of 7.9) and finally resuspended with 400 µL of the same buffer. When indicated, 15 µL of alkaline phosphatase from calf intestine (ALP) or 40 µL of 100 mM sodium orthovanadate (Na₃VO₄), as a phosphatase inhibitor, to a final concentration of 10 mM were added. Samples were incubated at 37 °C for 1 h and centrifugated at 3000 rpm for 1 min. We added 2× SDS loading buffer. Proteins were boiled for 5 min and then analyzed by SDS-PAGE and immunoblotting.