C7082

Lung tissues from each group were lysed with 100 μl/50 ml protein lysate RIPA. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 10% polyacrylamide gels were used for the separation of proteins. Then, the gels were transferred to polyvinylidenefluoride membranes. After blocking with 5% Skim milk/BSA, the membrane was incubated with primary antibodies (PPARγ, 1:1000, MAB3872, Chemicon International; PTEN, 1:1000, SAB4300337, Sigma; p-PTEN, pSer³⁸⁰/pThr³⁸²/pThr³⁸³, 1:1000, SAB4300044, Sigma; Akt, 1:1000, SAB4500796, Sigma; p-Akt, pSer¹²⁴, 1:1000, SAB4503853, Sigma; GSK-3β, 1:1000, G7914, Sigma; p-GSK-3β, pTyr²⁷⁹/pTyr²¹⁶, 1:1000, G5791, Sigma; β-catenin, 1:1000, C7082, Sigma; p-β-catenin, pSer³³/pSer, 1:1000, sc-57535, Santa Cruz Biotechnology), followed by treatment with HRP-labeled secondary antibody IgG (1:2000, sc-516102, Santa Cruz Biotechnology). The GAPDH (1:10000, sc-47724, Santa Cruz Biotechnology) was used as internal control. After treatment with ECL luminescence reagent (Thermo Scientific, U.S.A.), the relative gray value was analyzed using the Quantity one (Bio-Rad, U.S.A.).

Culture coverslips were fixed with 4% paraformaldehyde (Thermo Fisher) in PBS for 15 min. After 1 h in a blocking solution containing 0.25% Triton and 2% bovine serum albumin (BSA), cultures were incubated for 1 h at room temperature with primary antibodies as follows: rabbit anti-VE-cadherin (ab33168, Abcam), rabbit antiphospho myosin II light chain (36671s, Cell Signaling), mouse anti-paxillin (MA5-13356, Thermofisher), mouse anti-β-catenin (C7082, Sigma Aldrich), mouse anti-ZO1 (33-9100, Thermofisher), or rabbit anti-TGN46 (ab50595, Abcam). All antibodies were diluted 1/400–1/200 in a solution containing 0.25% Triton and 1% bovine serum albumin (BSA). Coverslips were washed three times with PBS and incubated for 1 h at room temperature with Alexa Fluor 555-conjugated donkey anti-rabbit antibody (ab150074, Abcam) or Alexa Fluor 488-conjugated donkey anti-mouse antibody (ab150105, Abcam) and DAPI. F-actin staining was performed using phalloidin (Sigma).